EP0338767A2 - Rekombinante und chimäre Antikörper gegen ein menschliches Adenocarcinoma-Antigen - Google Patents

Rekombinante und chimäre Antikörper gegen ein menschliches Adenocarcinoma-Antigen Download PDF

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Publication number
EP0338767A2
EP0338767A2 EP89303814A EP89303814A EP0338767A2 EP 0338767 A2 EP0338767 A2 EP 0338767A2 EP 89303814 A EP89303814 A EP 89303814A EP 89303814 A EP89303814 A EP 89303814A EP 0338767 A2 EP0338767 A2 EP 0338767A2
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Prior art keywords
plasmid
dna
fragment
host cell
restriction
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French (fr)
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EP0338767A3 (de
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Lisa Selsam Beavers
Thomas Frank Bumol
Robert Alan Gadski
Barbara Jean Weigel
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Eli Lilly and Co
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Eli Lilly and Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/461—Igs containing Ig-regions, -domains or -residues form different species
    • C07K16/462—Igs containing a variable region (Fv) from one specie and a constant region (Fc) from another
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/867—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof involving immunoglobulin or antibody produced via recombinant dna technology

Definitions

  • the present invention provides novel DNA compounds and recombinant DNA cloning vectors that encode monoclonal antibody KS1/4, as well as mouse/human chimeric antibodies derived from KS1/4.
  • the vectors allow expression of the novel DNA compounds in non-­lymphoid eukaryotic cells.
  • the present invention also provides host cells transformed with these novel cloning vectors.
  • the transformed host cells express the recombinant or chimeric KS1/4 antibodies, or deriv­atives thereof.
  • Many of the present DNA compounds can be used to produce KS1/4 derivatives never before synthesized either in nature or in the laboratory, and the present invention also comprises these unique molecules.
  • Monoclonal antibody KS1/4 is a murine anti­body which specifically binds to the ⁇ 40,000 dalton cell surface antigen found in high density on adeno­carcinoma cells and found also on normal epithelial cells. This antibody has been shown to be effective for the in vitro detection of disease, as well as the in vivo diagnosis and treatment of adenocarcinoma. Recent studies have confirmed that KS1/4-drug conjugates demon­strate a dose-dependent suppression of tumor growth. See, Spearman et al ., 1987, J. Pharmacol. and Exp. Therapeutics 241:695-703; Bumol et al ., in Ceriani, R.L. ed.
  • Chimeric antibodies comprise an antigen specific or variable region derived from one species joined with the con­stant region from a different species. See, Oi and Morrison, BioTechniques 4:214-221 (1986). Inasmuch as the immune response is often directed against the constant region, the replacement of a murine constant region with a human constant region will greatly diminish a patient's immunological reaction. Accordingly, chimeric antibodies are highly desirable for the treatment of disease.
  • the general concept of chimeric antibodies has been described, yet the development of novel chimeric antibodies having certain specificities is still needed.
  • the present invention discloses recombinant DNA and amino acid sequences which comprise the entire KS1/4 monoclonal antibody molecule. These sequences have been manipulated to express chimeric antibodies which have the same tissue specificity as KS1/4, but which comprise constant regions derived from human sources. The invention therefore will allow a therapeutic regimen with the same tissue specificity of monoclonal antibody KS1/4 but with greatly reduced immunological side effects.
  • the present invention further comprises the recombinant DNA and amino acid sequences of KS1/4.
  • the knowledge of these sequences allows skilled artisans to develop novel KS1/4 derivatives with modified affinity for the KS1/4 antigen.
  • the present invention further comprises methods of producing recombinant and chimeric KS1/4 in non-lymphoid cell lines, thereby circumventing the problems often arising from dual secretion of heterologous antibodies in lymphoid cells.
  • a - deoxyadenosine Ala - an alanine residue.
  • Ap R the ampicillin-resistant phenotype or gene conferring same.
  • Asn - an asparagine residue.
  • Asp an aspartic acid residue.
  • C deoxycytosine.
  • Chimeric antibody an antibody comprising a variable region from one species, typically mouse, joined to a constant region from a second and different species, typically human. Cys - a cysteine residue. dhfr - the dihydrofolate reductase phenotype or gene conferring same.
  • G deoxyguanosine.
  • G418 R the G418-resistant phenotype or gene conferring same. May also be identified as Km R .
  • Hm R the hygromycin-resistant phenotype or gene conferring same.
  • Ile an isoleucine residue.
  • KSA the ⁇ 40,000 dalton cell surface glyco-­protein antigen of UCLA-P3 cells that is recognized by monoclonal antibody KS1/4.
  • KS1/4 a murine monoclonal antibody derived from a hybridoma cell line, said antibody recognizing the ⁇ 40,000 dalton glycoprotein antigen found on the cell surface of P3-UCLA cells.
  • LP - a DNA segment comprising the promoter activity of the adenovirus late promoter. Lys - a lysine residue. Met - a methionine residue.
  • MoAB monoclonal antibody.
  • Nascent protein the polypeptide produced upon translation of a mRNA transcript, prior to any post-translational modifications.
  • pA - a DNA sequence encoding a polyadenylation signal.
  • Phe a phenylalanine residue.
  • pL a DNA segment comprising the promoter activity of the bacteriophage ⁇ leftward promoter.
  • Pro a proline residue.
  • Promoter a DNA sequence that directs transcription of DNA into RNA.
  • Recombinant DNA Cloning Vector any auto­nomously replicating agent, including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can be or have been added.
  • Recombinant DNA Expression Vector any re­combinant DNA cloning vector into which a promoter has been incorporated.
  • Replicon A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • Restriction Fragment any linear DNA sequence generated by the action of one or more restriction endonuclease enzymes.
  • Sensitive Host Cell - a host cell that cannot grow in the presence of a given antibiotic or other toxic compound without a DNA segment that confers resistance thereto.
  • Structural Gene any DNA sequence that encodes a functional polypeptide, inclusive of trans­lational start and stop signals. T - deoxythymidine.
  • Tc R the tetracycline-resistant phenotype or gene conferring same. Thr - a threonine residue. Trp - a tryptophane residue. Tyr - a tyrosine residue. Val - a valine residue.
  • the present invention is a recombinant DNA compound which comprises DNA encoding a monoclonal antibody light chain wherein the light chain is the light chain of monoclonal antibody KS1/4 and has an amino acid residue sequence substantially the same as:
  • the present invention is a recombinant DNA compound which comprises DNA encoding a monoclonal antibody heavy chain wherein the heavy chain is the heavy chain of monoclonal antibody KS1/4 and has an amino acid residue sequence substantially the same as:
  • the compounds of the present invention repre­sent recombinant monoclonal antibody KS1/4 and the heretofore unknown amino acid and nucleotide sequences of KS1/4. Due to the complementary nature of DNA base pairing, the sequence of one strand of a double-stranded DNA molecule is sufficient to determine the sequence of the opposing strand.
  • the nucleotide sequence of the light chain of KS1/4 is: while the nucleotide sequence of the heavy chain of KS1/4 is:
  • the present invention further comprises a recombinant DNA compound which comprises DNA encoding a chimeric antibody light chain comprising an antigen-­specific variable region derived from a first mammalian species and a constant region derived from a second and different mammalian species, said light chain variable region having an amino acid sequence substantially the same as:
  • the nucleotide sequence of the light chain variable region is:
  • the invention comprises derivatives of the light chain variable region disclosed above, one such derivative having an amino acid residue sequence substantially the same as:
  • nucleotide sequence of this light chain variable region derivative is:
  • the invention also comprises a recombinant DNA compound which comprises DNA encoding a chimeric antibody heavy chain variable region derived from a first mammalian species and a constant region derived from a second and different mammalian species, said heavy chain variable region having an amino acid sequence substantially the same as:
  • nucleotide sequence of this heavy chain variable region is:
  • Both the light chain and heavy chain molecules of the present invention are associated with distinct signal peptides.
  • the amino acid residue sequence of the light chain signal peptide is substantially the same as:
  • nucleotide sequence of this signal peptide is:
  • amino acid residue sequence of the heavy chain signal peptide is substantially the same as:
  • nucleotide sequence of this signal peptide is:
  • the novel DNA compounds of the present inven­tion are derived from cDNA clones prepared from the mRNA from the hybridoma cell line which makes monoclonal antibody KS1/4.
  • Plasmid pGKC2310 comprises the entire coding sequence of the light chain of monoclonal anti­body KS1/4, the coding sequence of the signal peptide associated with the light chain, and the 5′ and 3′ untranslated regions of this molecule.
  • the 5′ un­translated region has the DNA sequence: whereas the 3′ untranslated region has the DNA sequence:
  • Plasmid pGKC2310 can be conventionally isolated from E.
  • coli K12 MM294/pGKC2310 a strain deposited and made part of the permanent stock culture collection of the Northern Regional Research Laboratory (NRRL), Peoria, Illinois on April 1, 1988.
  • a culture of E. coli K12 MM294/pKC2310 can be obtained from the NRRL under the accession number B-18356.
  • a restriction site and function map of plasmid pKC2310 is presented in Figure 12 of the accompanying drawings.
  • Plasmid pG2A52 comprises the entire coding sequence of the heavy chain of monoclonal antibody KS1/4, the coding sequence of the signal peptide associated with the heavy chain, and the 5′ and 3′ untranslated regions of this molecule.
  • the heavy chain encoded by this molecule is of the IgG2A subclass.
  • the 5′ untranslated region has the DNA sequence: 5′-TCG TTT GTC TTA AGG CAC CAC TGA GCC CAA GTC TTA GAC ATC-3′ whereas the 3′ untranslated region has the DNA sequence: Plasmid pG2A52 can be conventionally isolated from E.
  • Plasmid CHKC2-6 comprises the cDNA coding sequence of the natural light chain variable region of monoclonal antibody KS1/4, the cDNA coding sequence of the signal peptide associated with the light chain, and a genomic DNA sequence which encodes the light chain constant region of a human immunoglobulin.
  • Plasmid CHKC2-6 can be conventionally isolated from E. coli K12 DH5/CHKC2-6, also deposited and made part of the permanent stock culture collection of the NRRL on April 1, 1988.
  • a cul­ture of E. coli K12 DH5/CHKC2-6 can be obtained from the NRRL under the accession number NRRL B-18358.
  • a restriction site and function map of plasmid CHKC2-6 is presented in Figure 13 of the accompanying drawings.
  • Plasmid CHKC2-18 comprises the cDNA coding sequence of a derivative light chain variable region of monoclonal antibody KS1/4, the cDNA coding sequence of the signal peptide associated with the light chain, and a genomic DNA sequence which encodes the light chain constant region of a human immunoglobulin.
  • the variation in this sequence comprises the alteration of the codon at the 3′ terminus.
  • the natural light chain variable region contains an arginine codon at the 3′ terminus, whereas the codon in the same position in plasmid CHKC2-18 encodes a glycine residue.
  • Plasmid CHKC2-18 can be conventionally isolated from E.
  • E. coli K12 DH5/CHKC2-18 also deposited and made part of the permanent stock culture collection of the NRRL on April 1, 1988.
  • a culture of E. coli K12 DH5/CHKC2-18 can be obtained from the NRRL under the accession number NRRL B-18359.
  • a restriction site and function map of plasmid CHKC2-18 is presented in Figure 13 of the accompanying drawings.
  • Plasmid CH2A5 comprises the cDNA coding sequence of the heavy chain variable region of mono­clonal antibody KS1/4, the cDNA coding sequence of the signal peptide associated with said heavy chain, and a genomic DNA sequence which encodes the heavy chain constant region of human immunoglobulin IgG1.
  • Plasmid CH2A5 can be conventionally isolated from E. coli K12 MM294/CH2A5, also deposited and made part of the per­manent stock culture collection of the NRRL on April 1, 1988.
  • a culture of E. coli K12 MM294/CH2A5 can be obtained from the NRRL under the accession number NRRL B-18360.
  • a restriction site and function map of plasmid CH2A5 is presented in Figure 14 of the accompanying drawings.
  • Plasmid CH2A5IG2 comprises the cDNA coding sequence of the heavy chain variable region of mono­clonal antibody KS1/4, the cDNA coding sequence of the signal peptide associated with the heavy chain and a genomic DNA sequence which encodes the heavy chain constant region of human immunoglobulin IgG2.
  • Plasmid CH2A5IG2 can be conventionally isolated from E. coli K12 DH5/CH2A5IG2, also deposited and made part of the permanent stock culture collection of the NRRL on April 1, 1988.
  • a culture of E. coli K12 DH5/CH2A5IG2 can be obtained from the NRRL under the accession number NRRL B-18361.
  • a restriction site and function map of plasmid CH2A5IG2 is presented in Figure 14 of the accompanying drawings.
  • Plasmid CH2A5IG3 comprises the cDNA coding sequence of the heavy chain variable region of mono­clonal antibody KS1/4, the cDNA coding sequence of the signal peptide associated with the heavy chain and a genomic DNA sequence which encodes the heavy chain constant region of human immunoglobulin IgG3.
  • Plasmid CH2A5IG3 can be conventionally isolated from E. coli K12 DH5/CH2A5IG3, also deposited and made part of the permanent stock culture collection of the NRRL on April 1, 1988.
  • a culture of E. coli K12 DH5/CH2A5IG3 can be obtained from the NRRL under the accession number NRRL B-18362.
  • a restriction site and function map of plasmid CH2A5IG3 is presented in Figure 15 of the accompanying drawings.
  • Plasmid CH2A5IG4 comprises the cDNA coding sequence of the heavy chain variable region of mono­clonal antibody KS1/4, the cDNA coding sequence of the signal peptide associated with the heavy chain and a genomic DNA sequence which encodes the heavy chain constant region of human immunoglobulin IgG4.
  • Plasmid CH2A5IG4 can be conventionally isolated from E. coli K12 DH5/CH2A5IG4, also deposited and made part of the permanent stock culture collection of the NRRL on April 1, 1988.
  • a culture of E. coli K12 DH5/CH2A5IG4 can be obtained from the NRRL under the accession number NRRL B-18363.
  • a restriction site and function map of plasmid CH2A5IG4 is presented in Figure 15 of the accompanying drawings.
  • the present DNA compounds which encode recombinant KS1/4 and derivatives are especially pre­ferred for the construction of vectors for transfor­mation and expression of the various antibody chains in mammalian and other eukaryotic cells.
  • Many mam­malian host cells possess the necessary cellular machinery for the recognition and proper processing of the signal peptides present on the amino-terminus of the various antibody chains embodied in the present invention.
  • Some mammalian host cells also provide the post-translational modifications, such as glycosylation, that are observed in antibody molecules.
  • a wide variety of vectors exist for the transformation of eukaryotic host cells, and the specific vectors exemplified below are in no way intended to limit the scope of the present invention.
  • Plasmid phd was constructed from a wide variety of publicly available starting materials. A restriction site and function map of plasmid phd is presented in Figure 11 of the accompanying drawings.
  • Plasmid phd was constructed by first isolating plasmid pKC283 from E. coli K12 BE1201/pKC283. This culture may be obtained from the NRRL under accession number NRRL B-15830 (deposited August 3, 1984). Plasmid pKC283 comprises a hybrid lpp -pL promoter of bacterio­phage ⁇ . This plasmid is obtained from E. coli K12 BE1201 cells because these cells comprise a temperature sensitive cI repressor integrated into the cellular DNA. The unneeded lac Z portion of plasmid pKC283 was excised by first digesting the plasmid with restriction enzyme Pvu II.
  • Plasmid pKC283PX was next digested with restriction enzymes Bgl II and Xho I. After the vector was purified, DNA linkers with Bgl II and Xho I ends were ligated into the vector to form plasmid pKC283-L.
  • the Bgl II- Xho I linker also contained an Xba I site.
  • the Xho I site of plasmid pKC283-L was next converted into a Bam HI site. This was accomplished by a total digestion of plasmid pKC283-L with restriction enzyme Xho I, followed by treatment with Klenow, then addition of Bam HI linkers, to form plasmid pKC283-LB.
  • E. coli DNA was next excised from plasmid pKC283PX by total digestion with restric­tion enzyme Sal I, followed by treatment of the ⁇ 4.0 kb vector with Klenow, then addition of Eco RI linkers. Upon recircularization via ligation, this formed plasmid pKC283PRS. Plasmid pKC283PRS was then digested with restriction enzymes Pst I and Sph I and the ⁇ 0.85 kb Pst I- Sph I restriction fragment was isolated. In an analogous manner, plasmid pKC283-LB was digested with restriction enzymes Pst I and Sph I and the ⁇ 3.0 kb frag­ment was isolated.
  • Plasmid pNM789 was partially digested with restriction enzyme Pvu II, fully digested with restriction enzyme Bam HI, then treated with alkaline phosphatase. Next, a new Pvu II- Bam HI linker was ligated into the digested, phosphatased vector pNM789 to form plasmid 120.
  • Plasmid 120 was then totally digested with restriction enzymes Xba I and Bam HI and the ⁇ 0.6 kb Xba I- Bam HI EK-BGH-encoding restriction fragment was isolated.
  • Plasmid pL32 was also digested with restriction enzymes Xba I and Bam HI and the ⁇ 3.9 kb vector fragment was isolated.
  • the ⁇ 0.6 kb Xba I- Bam HI fragment of plasmid 120 was then ligated into the ⁇ 3.9 kb vector fragment of plasmid pL32 to form plasmid pL47.
  • Detailed descriptions of the construction of plasmids 120 and pL47 are presented in Examples 6 and 7. Restriction site and function maps of plasmids 120 and pL47 are presented respectively in Figures 3 and 4 of the accompanying drawings.
  • Plasmid pPR12 comprises the temperature-­sensitive pL repressor gene cI857 and the plasmid pBR322 tetracycline resistance-conferring gene. Plasmid pPR12 is disclosed and claimed in U.S. Patent No. 4,436,815, issued 13 March, 1984. A restriction site and function map of plasmid pPR12 is presented in Figure 4 of the accompanying drawings. The Eco RI site was removed from plasmid pPR12 by first totally digesting the plasmid with restriction enzyme Eco RI, followed by treatment with Klenow. The vector was then recircularized by ligation to form plasmid pBRI2 ⁇ R1.
  • Plasmid pPR12 ⁇ R1 was then digested with restriction enzyme Ava I and treated with Klenow.
  • the Ava I-digested, Klenow treated pPR12 ⁇ R1 was next ligated to Eco RI linkers, cut with restriction enzyme Eco RI, then recircularized to form plasmid pPR12AR1.
  • a detailed description of the con­struction of plasmid pPR12AR1 is presented in Example 8.
  • a restriction site and function map of plasmid pPR12AR1 is presented in Figure 4 of the accompanying drawings.
  • Plasmid pL47 was digested with restriction enzymes Pst I and Bam HI and the ⁇ 2.7 kb Pst I- Bam HI restriction fragment was isolated.
  • plasmid pL47 was digested with restriction enzymes Eco RI and Bam HI and the ⁇ 1.03 kb Eco RI- Bam HI fragment was iso­lated.
  • the BK enhancer-type vector of the present invention comprises a BK enhancer-adenovirus late promoter cassette plus a hygromycin resistance con­ferring gene and a murine dihydrofolate reductase (dhfr) gene.
  • the use of the BK virus enhancer in conjunction with the adenovirus late promoter significantly in­creases transcription of a recombinant gene in eukaryotic host cells.
  • the hygromycin resistance-­conferring gene is present as a selectable marker for use in eukaryotic host cells.
  • the murine dihydrofolate reductase gene under appropriate conditions, is amplified in the host chromosome.
  • dhfr gene is a selectable marker in dhfr-negative cells and can be used to increase the copy number of a DNA segment by exposing the host cell to increasing levels of methotrexate.
  • Plasmid pLPChd may be used to construct a eukaryotic expression vector for expression of the novel KS1/4 of the present invention.
  • Plasmid pLPChd contains the dhfr gene, the Adenovirus type-2 promoter and the BK virus enhancer.
  • the BK virus which contains the BK virus enhancer, can be purchased or readily isolated in large quantities as described in Example 10.
  • the BK virus is also available from the American Type Culture Collection under the accession number ATCC VR-837. A restriction site and function map of the BK virus is presented in Figure 5 of the accompanying drawings.
  • Plasmid pdBPV-MMTneo comprises the replicon and ⁇ -lactamase gene from plasmid pBR322, the mouse metallothionein promoter positioned to drive expression of a structural gene that encodes a neomycin resistance-conferring enzyme, and about 8 kb of bovine papilloma virus (BPV) DNA.
  • Plasmid pdBPV-MMTneo can be digested with restric­tion enzyme Bam HI to generate two fragments: the ⁇ 8 kb fragment that comprises the BPV DNA and an ⁇ 7 kb frag­ment that comprises the other sequences described above.
  • BK virus has only one Bam HI restriction site, and plasmids pBKneo1 and pBKneo2 were constructed by ligating the ⁇ 7 kb Bam HI restriction fragment of plasmid pdBPV-MMTneo to Bam HI-linearized BK virus DNA.
  • Plasmid pBKneo1 contains an ⁇ 2.1 kb Sal I- Hin dIII restriction fragment, whereas plasmid pBKneo2 contains an ⁇ 1.0 kb restriction fragment.
  • a restriction site and function map of plasmid pBKneo1 is presented in Figure 5 of the accompanying drawings.
  • Plasmids pBKneo1 and pBKneo2 each comprise the entire genome of the BK virus, including the enhancer sequence, and thus serve as useful starting materials for the expression vector of the present invention.
  • Expression vector, plasmid pBLcat comprises the BK enhancer sequence in tandem with the human adenovirus­type-2 late promoter positioned to drive expression of the chloramphenicol acetyltransferase enzyme (CAT).
  • Plasmid pSV2cat serves as a convenient source of the CAT gene and can be obtained from the ATCC under the accession number ATCC 37155.
  • Human adenovirus-type-2 DNA is commercially available and can also be obtained from the ATCC under the accession number ATCC VR-2.
  • Illustrative plasmid pBLcat was constructed by ligating the ⁇ 0.32 kb late-promoter-containing Acc I- Pvu II restriction fragment of human adenovirus-type-2 DNA to blunt-ended Bcl I linkers that attached only to the Pvu II end of the Acc I- Pvu II restriction fragment. The resulting fragment was then ligated to the ⁇ 4.51 kb Acc I- Stu I restriction fragment of plasmid pSV2cat to yield intermediate plasmid pLPcat.
  • the desired plasmid pBLcat was constructed from plasmid pLPcat by ligating the origin of replication and enhancer-containing ⁇ 1.28 kb Acc I- Pvu II restriction fragment of BK virus DNA to the ⁇ 4.81 kb Acc I- Stu I restriction fragment of plasmid pLPcat.
  • the construction of plasmid pBLcat is further described in Example 12. Restriction site and function maps of plasmids pLPcat and pBLcat are pre­sented in Figure 6 of the accompanying drawings.
  • Plasmid pL133 was next constructed from plasmids pHC7, pSV2gpt and pSV2- ⁇ -globin.
  • Plasmid pHC7 comprises a DNA sequence which encodes human protein C.
  • Plasmid pHC7 can be isolated from E. coli K12 RR1/pHC7 which is available from the NRRL under accession number NRRL B-15926 (deposited January 29, 1985).
  • Plasmid pHC7 was cut with restriction enzyme Ban I and the ⁇ 1.25 kb restriction fragment was isolated. Linkers were added, and the fragment was then cut with restriction enzymes Apa I and Hin dIII, then the desired ⁇ 1.23 kb restriction fragment was isolated.
  • Plasmid pHC7 was next cut with restriction enzyme Pst I, the ⁇ 0.88 kb restriction fragment was isolated, linkers were added, the fragment was re-cut with restriction enzymes Apa I and Bgl II and the ⁇ 0.19 kb Apa I- Bgl II restriction fragment was isolated.
  • Plasmid pSV2gpt (ATCC 37145) was digested with restriction enzymes Hin dIII and Bgl II and the ⁇ 5.1 kb fragment was isolated.
  • Plasmid pSV2-HPC8 was then cut with restric­tion enzymes Hin dIII and Sal I, and the ⁇ 0.29 kb re­striction fragment was isolated. Likewise, plasmid pSV2-HPC8 was also cut with restriction enzymes Bgl II and Sal I, and the ⁇ 1.15 kb restriction fragment was isolated. Plasmid pSV2- ⁇ -globin (NRRL B-15928, deposited January 29, 1985) was cut with restriction enzymes Bgl II and Hin dIII and the ⁇ 4.2 kb restriction fragment was isolated. These three fragments were then ligated together to form plasmid pL133.
  • Plasmid pL133 was digested with restriction enzyme Hin dIII, then treated with alkaline phosphatase. Plasmid pBLcat was also cut with restriction enzyme Hin dIII and the ⁇ 0.87 kb restriction fragment was isolated. This fragment was ligated into the Hin dIII cut, phosphatased plasmid pL133 vector to form plasmid pLPC. Because the Hin dIII fragment of plasmid pBLcat can be inserted into plasmid pL133 in two orientations, it should be noted that pLPC is the plasmid wherein the proper orientation provides an ⁇ 1.0 kb Nde I- Stu I fragment.
  • Plasmid pLPC like plasmid pL133, comprises the enhancer, early and late promoters, T-antigen-binding sites, and origin of replication of SV40.
  • Detailed protocols for the con­struction of plasmids pL133 and pLPC are provided in Examples 13 and 14. Restriction site and function maps of plasmids pL133 and pLPC are presented respectively in Figures 7 and 8 of the accompanying drawings.
  • the SV40 elements present on plasmid pLPC are situated closely together and are difficult to delineate.
  • the binding of T antigen to the T-antigen-­binding sites, which is necessary for SV40 replication, is known to enhance transcription from the SV40 late promoter and surprisingly has a similar effect on the BK late promoter.
  • plasmid pLPC nor plasmid pL133 are stably maintained as episomal (extrachromosomal) elements in the presence of SV40 T antigen, but rather, the two plasmids must integrate into the chromosomal DNA of the host cell to be stably maintained.
  • the overall structure of the BK enhancer region is quite similar to that of SV40, for the BK enhancer, origin of replication, early and late promoters, and the BK analogue of the T-antigen-­binding sites are also closely situated and thus dif­ficult to delineate on the BK viral DNA.
  • a plasmid that comprises the BK origin of replication and T-antigen-­binding sites does not replicate to an extent that proves lethal and is stably maintained as an episomal element in the host cell.
  • the T-antigen-­driven replication can be used to increase the copy number of a vector comprising the BK origin of repli­cation so that when selective pressure is applied more copies of the plasmid integrate into the host cell's chromosomal DNA.
  • BK replication is also stimulated by SV40 T antigen.
  • Episomal maintenance of a recombinant DNA expression vector is not always preferred over integra­tion into the host cell chromosome.
  • plasmid pLPC due to the absence of a selectable marker that functions in eukaryotic cells, the identification of stable, eukaryotic transformants of plasmid pLPC is difficult, unless plasmid pLPC is cotransformed with another plasmid that does comprise a selectable marker.
  • plasmid pLPC has been modified to produce derivative plasmids that are selectable in eukaryotic host cells.
  • Plasmid pSV2hyg can be obtained from the Northern Regional Research Laboratory (NRRL), Peoria, IL 61640, under the accession number NRRL B-18039 (deposited February 11, 1986).
  • NRRL Northern Regional Research Laboratory
  • Peoria Peoria
  • IL 61640 a plasmid that comprises a hygromycin resistance-conferring gene.
  • NRRL B-18039 accession number NRRL B-18039
  • Plasmid pSV2hyg was digested with restriction enzyme Bam HI, and the ⁇ 2.5 kb Bam HI restriction frag­ment, which comprises the entire hygromycin resistance-­conferring gene, was isolated, treated with Klenow enzyme (the large fragment produced upon subtilisin cleavage of E. coli DNA polymerase I), and then ligated to the Klenow-treated, ⁇ 5.82 kb Nde I- Stu I restriction fragment of plasmid pLPC to yield plasmids pLPChyg1 and pLPChyg2. Plasmids pLPChyg1 and pLPChyg2 differ only with respect to the orientation of the hygromycin resistance-conferring fragment.
  • Plasmid pLPChyg1 contains an ⁇ 5.0 kb Hin dIII fragment whereas plasmid pLPChyg2 contains an ⁇ 1.0 kb fragment.
  • the construction protocol for plasmids pLPChyg1 and pLPChyg2 is described in Example 15.
  • a restriction site and function map of plasmid pLPChyg1 is presented in Figure 8 of the accompanying drawings.
  • Plasmid pBW32 which contains the murine dihydrofolate reductase (dhfr) gene, was constructed next.
  • Plasmid pTPA102 (NRRL B-15834, deposited August 10, 1984) was cut with restriction enzyme Tth 111I and the ⁇ 4.4 kb restriction fragment was isolated. This fragment was treated with Klenow, linkers were added, then the fragment was cut with restriction enzymes Hin dIII and Bam HI to yield an ⁇ 2.0 kb restriction fragment.
  • Plasmid pRC was then constructed by ligating the ⁇ 288 bp Cla I- Eco RI restriction fragment of pTPA102 into Cla I- Eco RI cut vector pKC7.
  • Plasmid pKC7 can be obtained from the ATCC under the accession number ATCC 37084. Plasmid pRC was digested with restriction enzymes Bam HI and Hin dIII, then ligated to the ⁇ 2.0 kb restriction fragment of plasmid pTPA102, formed above, to yield plasmid pTPA103. The construction protocol for plasmid pTPA103 is described in Example 16A. A schematic of this construction is presented in Figure 9 of the accompanying drawings.
  • Plasmid pTPA103 was cut with restriction enzyme Bgl II, treated with Klenow, and the Nde I linkers were added. This mixture was then ligated to form plasmid pTPA103der Nde I. Plasmid pTPA103der Nde I was cut with restriction enzyme Ava II, and the ⁇ 1.4 kb fragment was isolated. This fragment was treated with Klenow, then, after the addition of Hpa I linkers, was cut with restriction enzyme Eco RI. The ⁇ 770 bp frag­ment, containing trp PO and the amino terminus of TPA, was ligated into Eco RI- Sma I digested vector pUC19, to form pUC19TPAFE.
  • Plasmid pUC19TPAFE was partially digested with restriction enzyme Hpa I, then totally cut with restriction enzyme Bam HI. The resultant ⁇ 3.42 kb Hpa I- Bam HI restriction fragment was then ligated to the ⁇ 1.015 Sca I- Bam HI fragment derived from plasmid pTPA103 to form plasmid pBW25.
  • the construction protocol for plasmid pBW25 is described in Example 16B. A schematic of this construction is presented in Figure 9 of the accompanying drawings.
  • Plasmid pBW25 was cut with restriction enzymes Hin dIII and Eco RI and the resultant ⁇ 810 bp fragment was ligated into Hin dIII- Eco RI cut phage M13mp8 (New England Biolabs) to form phage pM8BW26.
  • An in vitro mutagenesis reaction was then performed on phage pM8BW26 (deleting DNA coding for amino acid residues) to form phage pM8BW27.
  • Phage pM8BW27 was cut with restriction enzymes Eco RI and Nde I and the ⁇ 560 bp restriction fragment was isolated.
  • a synthetic Nde I-­ Xba I linker of ⁇ 48 bp was synthesized.
  • Plasmid pTPA103 was cut with restriction enzymes Eco RI and Bam HI and the ⁇ 689 bp fragment was isolated.
  • Plasmid pL110 (constructed in Example 9) was partially digested with restriction enzyme Bam HI, then totally cut with Xba I and the ⁇ 6.0 kb fragment was isolated.
  • This ⁇ 6.0 kb vector fragment, the ⁇ 689 bp fragment of plasmid pTPA103, the ⁇ 560 bp fragment of phage pM8BW27, and the ⁇ 48 bp linker were all then ligated together to form plasmid pBW28.
  • the construction protocol of plasmid pBW28 is described in Example 16C. A schematic of this construction is presented in Figure 10 of the accompanying drawings.
  • Plasmid pTPA301 was next formed by ligating the ⁇ 2.0 kb Hin dIII- Bgl II fragment of plasmid pTPA103 to the ⁇ 4.2 kb Hin dIII- Bgl II fragment of plasmid pSV2- ⁇ -globin. Plasmid pSV2-dhfr (ATCC 37146) was cut with restriction enzyme Pvu II. Following the addition of Bam HI linkers, the ⁇ 1.9 kb dhfr gene-containing fragment was ligated into Bam HI cut, phosphatased plasmid pTPA301 to form plasmid pTPA303.
  • Plasmid pTPA301 was cut with restriction enzymes Eco RI and Bgl II to yield an ⁇ 2.7 kb fragment.
  • Plasmid pTPA303 was cut with restriction enzymes Hin dIII and Eco RI to yield the ⁇ 2340 bp dhfr gene containing fragment.
  • Plasmid pTPA303 was cut with restriction enzymes Hin dIII and Sst I to yield an ⁇ 1.7 kb fragment.
  • Plasmid pBW28 was cut with restriction enzymes Xho II and Sst I to yield an ⁇ 680 bp fragment.
  • Plasmid pLPChyg1 contains two Eco RI restriction enzyme recognition sites, one in the hygromycin resistance-conferring gene and one in the plasmid pBR322-derived sequences.
  • plasmid pLPChd1 has been designated plasmid pLPChd.
  • a restriction site and function map of plasmid pLPChd is presented in Figure 11 of the accompanying drawings. The construction of plasmids pLPChd1 and pLPChd2, which differ only with respect to the orientation of the dhfr gene-containing DNA segment, is described in Example 17.
  • Plasmid phd was next constructed by trans­forming and re-isolating plasmid pLPChd through a dam ⁇ strain of E. coli . Plasmid pLPChd was then digested with restriction enzyme Bcl I and recircularized to form plasmid phd. Plasmid phd results from the deletion of the extra Bcl I linkers that attached during the con­struction of plasmid pLPcat and the two adjacent Bcl I restriction fragments of a total size of about 1.45 kb from plasmid pLPChd.
  • the DNA encoding the full length cDNA which encodes the light chain of monoclonal antibody KS1/4 is ligated into plasmid phd to form expression vector pL-KSL.
  • the ⁇ 1100 base pair Eco RI fragment of plasmid pGKC2310 is treated with Klenow, ligated to Bam HI linkers, then ligated into Bcl I-­digested plasmid phd.
  • Detailed descriptions of the construction of plasmids phd and pL-KSL are provided in Examples 18 and 19.
  • a restriction site and function map of plasmid phd is presented in Figure 11 of the accompanying drawings.
  • Plasmid pH-KS is a vector of the present invention derived from plasmid phd and plasmid pG2A52.
  • the ⁇ 1.6 kb Eco RI fragment of plasmid pG2A52 is iso­lated, treated with Klenow and joined to Bam HI linkers.
  • This fragment comprises the full length cDNA which encodes the heavy chain of monoclonal antibody KS1/4.
  • This fragment is next ligated into Bcl I-digested plasmid phd to form expression plasmid pH-KS.
  • Plasmid pL-HD is an expression vector constructed from plasmid phd and plasmid CHKC2-18, which contains DNA encoding a deriv­ative of the KS1/4 light chain variable region joined to a human light chain constant region.
  • the ⁇ 1.3 kb Fnu DII- Hin dIII fragment of plasmid CHKC2-18 was isolated and treated with Klenow. This fragment was then ligated into Bcl I-digested, Klenow treated plasmid phd to form expression plasmid pL-HD.
  • Detailed descriptions of the construction of plasmids pH-KS and pL-HD are presented respectively in Examples 20 and 21.
  • Plasmid pL-HD2 is an expression vector con­structed from plasmid phd and plasmid CHKC2-6, which contains DNA encoding the natural sequence of the KS1/4 light chain variable region joined to a human light chain constant region.
  • the ⁇ 1.3 kb Fnu DII- Hin dIII fragment of plasmid CHKC2-18 was isolated and treated with Klenow. This fragment was then ligated into Bcl I-digested, Klenow-treated plasmid phd to form expression plasmid pL-HD2.
  • Plasmid pH1-HD which contains cDNA encoding the heavy chain variable region of KS1/4 joined to genomic DNA encoding a human IgG1 constant region, was constructed from plasmid phd and plasmid CH2A5. Plasmid CH2A5 was digested with re­striction enzyme Eco RI, treated with Klenow, then ligated to Bam HI linkers. Next, the plasmid was digested with restriction enzyme Bam HI and the ⁇ 7.4 kb restriction fragment was isolated. This fragment was ligated into Bcl I-digested plasmid phd to form expression plasmid pH1-HD. Detailed descriptions of the construction of plasmids pL-HD2 and pH1-HD are presented respectively in Examples 22 and 23.
  • Plasmid pH2-HD which contains cDNA encoding the heavy chain variable region of KS1/4 joined to genomic DNA encoding a human IgG2 constant region, was constructed from plasmid phd and plasmid CH2A5IG2. Plasmid CH2A5IG2 was digested with restriction enzyme Eco RI, treated with Klenow, then ligated to Bam HI linkers. Next, the plasmid was digested with restric­tion enzyme Bam HI and the ⁇ 6.1 kb restriction fragment was isolated. This fragment was ligated into Bcl I-­digested plasmid phd to form expression plasmid pH2-HD.
  • Plasmid pH3-HD which contains cDNA encoding the heavy chain variable region of KS1/4 joined to genomic DNA encoding a human IgG3 constant region, was constructed from plasmid phd and plasmid CH2A5IG3. Plasmid CH2A5IG3 was digested with restriction enzyme Eco RI, treated with Klenow, then ligated to Bam HI linkers. Next, the plasmid was digested with restriction enzyme Bam HI and the ⁇ 7.4 kb restriction fragment was isolated. This fragment was ligated into Bcl I-digested plasmid phd to form expression plasmid pH3-HD. Detailed descriptions of the construction of plasmids pH2-HD and pH3-HD are presented respectively in Examples 24 and 25.
  • Plasmid pH4-HD which contains cDNA encoding the heavy chain variable region of KS1/4 joined to genomic DNA encoding a human IgG4 constant region, was constructed from plasmid phd and plasmid CH2A5IG4. Plasmid CH2A5IG4 was digested with restriction enzyme Eco RI, treated with Klenow, then ligated to Bam HI linkers. Next, the plasmid was digested with restric­tion enzyme Bam HI and the ⁇ 6.4 kb restriction fragment was isolated. This fragment was ligated into Bcl I-­digested plasmid phd to form expression plasmid pH4-HD. A more detailed description of the construction is presented in Example 26.
  • the present invention is in no way limited to the use of the particular eukaryotic promoters exempli­fied herein.
  • Other promoters such as homologous or heterologous immunoglobulin promoters, the SV40 late promoter or promoters from eukaryotic genes, such as for example, the estrogen-inducible chicken ovalbumin gene, the interferon genes, the glucocorticoid-inducible tyrosine aminotransferase gene, the thymidine kinase gene, the major early adenovirus gene, and the SV40 early promoter, can be readily isolated and modified for use on recombinant DNA expression vectors designed to produce antibodies in eukaryotic host cells.
  • Eukaryotic promoters can also be used in tandem to drive expression of such antibodies.
  • retroviruses are known that infect a wide range of eukaryotic host cells.
  • Long terminal repeats in the retrovirus DNA often encode promoter activity and can be used, in place of the BK enhancer-adenovirus late promoter described above, to drive expression of KS1/4 and its derivatives.
  • KS1/4 and its derivatives Neither is the present invention limited to the actual selectable marker exemplified on the plasmids exemplified herein.
  • selectable markers exist, both for eukaryotic and prokaryotic host cells, that are suitable for use on a recombinant DNA cloning or expression vector comprising a DNA compound (or sequence) of the present invention.
  • the various expression vectors can be trans­formed into and expressed in a variety of eukaryotic, especially mammalian, host cells.
  • the expression vectors also comprise sequences that allow for repli­cation in E. coli , as it is usually more efficient to prepare plasmid DNA in E. coli than in other host cells.
  • Expression of antibodies occurs in host cells in which the particular promoter associated with the antibody's structural gene functions. Skilled artisans will understand that a variety of eukaryotic host cells can be used to express the various antibodies using the BK enhancer-adenovirus late promoter, so long as the host cell expresses an immediate-early gene product of a large DNA virus.
  • the immediate-early gene product can be introduced into host cells by many means, such as transformation with a plasmid or other vector, virtually any eukaryotic cell can be used in the present method.
  • Human cells are preferred host cells in the method of the present invention, because human cells are the natural host for BK virus and may contain cellular factors that serve to stimulate the BK enhancer. While human cells may be used as host cells, the adenovirus 12-transformed Syrian Hamster cell line AV12, which expresses the E1A gene product, is most preferred and is available from the American Type Culture Collection in Rockville, Maryland, under the accession number ATCC CRL 9595 (deposited November 24, 1987). The normal transformation procedure is described in detail in Example 27.
  • AV12 cells When transformed with an expression vector that encodes an immunoglobulin heavy chain, AV12 cells secrete the processed heavy chain into the supernatant. However, AV12 cells transformed with an expression vector which encodes a light chain do not secrete said light chain unless the cells are also co-transformed with an expression vector encoding a heavy chain.
  • a procedure for isolating clones which express the various immunoglobulin chains of the present invention is pre­sented in Example 28.
  • Functional KS1/4 and KS1/4 derivatives can therefore be purified from the super­natant of AV12 cells which have been co-transformed with vectors encoding light and heavy chains. In this manner, many variants of KS1/4 can be produced by mixing and matching the different light and heavy chains of the present invention.
  • lymphoid systems such as myeloma or hybridoma cells.
  • lymphoid systems such as myeloma or hybridoma cells.
  • lymphoid cells When transformed with DNA encoding a separate antibody, or fused with other cells which produce a distinct antibody, such lymphoid cells some­times become dual secreters. Dual secreting cells lines then produce many antibodies with hybrid molecular structures, a large population of which are not desired. These unwanted hybrids must then be separated from the intended product using costly and time consuming techniques.
  • the method of the present invention traverses this problem in that non-lymphoid cells do not naturally secrete antibody molecules. Therefore, the only antibody which is secreted into the supernatant is the homogeneous product desired.
  • DNA sequences and plasmids are possible.
  • the degeneracy of the genetic code allows for the substitution of nucleotides through­out polypeptide coding regions as well as for the sub­stitution of the translational stop signal for the translational stop signals specifically exemplified.
  • sequences can be deduced from the now-known amino acid or DNA sequence of KS1/4 and can be constructed by following conventional synthetic proc­edures. Such synthetic methods can be carried out in substantial accordance with the procedures of Itakura et al ., 1977 Science 198:1056 and Crea et al ., 1978, Proc. Nat. Acad. Sci. USA 75:5765.
  • syn­thetic genes and linkers can be synthesized either by using a Systec 1450A DNA synthesizer (Systec Inc., 3816 Chandler Drive, Minneapolis, MN) or an ABS 380A DNA synthesizer (Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404).
  • Systec 1450A DNA synthesizer Systec Inc., 3816 Chandler Drive, Minneapolis, MN
  • ABS 380A DNA synthesizer Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404.
  • Many other DNA synthesizing instruments are known in the art and can be used to make synthetic DNA fragments. Therefore, the present invention is in no way limited to the DNA sequences and plasmids specifically exemplified.
  • the expression vectors of this invention are used to transform eukaryotic host cells, such that polypeptides with various light and heavy chain structures are expressed by the host cell. If the host cell is transformed with a vector comprising a promoter that functions in the host cell and drives transcription of the such immunoglobulin structural genes, and if the host cell possesses the cellular machinery with which to process the signal peptides, mature antibodies or antibody chains are secreted by such cells. Under other expression conditions, such as when only immunoglobulin light chains are expressed by the host cell, the light chains must be isolated from the host cell.
  • Monoclonal antibody KS1/4 has been shown to be an effective agent for the diagnosis, prognosis and treat­ment of adenocarcinoma by Bumol in Reisfeld, R.A. and Sell, S. eds. Monoclonal Antibodies and Cancer Therapy. New York: Alan R. Liss, Inc., 1985, 257-259. Spearman et al ., 1987, J. Pharmacol. and Exp.
  • Therapeutics 241:695-703 the teaching of which is herein incor­porated by reference, disclosed the use of a monoclonal antibody-vinca alkaloid conjugate in the localization and treatment of tumors.
  • This KS1/4-DAVLB(4-desacetyl­vinblastine) conjugate was also responsible for tumor growth suppression as disclosed by Bumol et al , in Ceriani, R.L. ed. Immunologic Approaches to the Diagnosis and Therapy of Breast Cancer, New York and London: Plenum Press; 1987, 205-215, the teaching of which is herein incorporated by reference.
  • Biochemical and immunological studies have revealed that the recombinant and chimeric KS1/4 molecules of the present invention possess the same antigen reactivity as KS1/4 molecules derived from hybridoma cells.
  • KS1/4 can be used to create novel, high or low affinity derivatives.
  • Various portions of the antibody may be deleted or mutated to create new antibodies, or portions of one chain may be replaced with a piece of another chain.
  • X-Ray crystallographic studies will demonstrate which amino acid residues of the antibody appear in close proximity to amino acid residues of the antigen to which KS1/4 binds.
  • KS1/4 can be modified to provide negative residues near positive residues on the antigen.
  • Such "engineered” antibodies will then display modified affinity to the cell surface antigen in cancer patients.
  • Lyophils of E. coli K12 BE1201/pKC283 are obtained from the Northern Regional Research Laboratory, Peoria, Illinois 61604, under the accession number NRRL B-15830.
  • the lyophils are decanted into tubes con­taining 10 ml LB medium (10 g Bacto-tryptone, 5 g Bacto-yeast extract, and 10 g NaCl per liter; pH is adjusted to 7.5) and incubated two hours at 32°C, at which time the cultures are made 50 ⁇ g/ml in ampicillin and then incubated at 32°C overnight.
  • 10 ml LB medium 10 g Bacto-tryptone, 5 g Bacto-yeast extract, and 10 g NaCl per liter; pH is adjusted to 7.5
  • coli K12 BE1201/pKC283 cells were cultured at 32°C, because the cells comprise a temperature-sensitive cI repressor gene integrated into the cellular DNA.
  • the temperature of incubation is 37°C.
  • a small portion of the overnight culture is placed on LB-agar (LB medium with 15 g/l Bacto-agar) plates containing 50 ⁇ g/ml ampicillin in a manner so as to obtain a single colony isolate of E. coli K12 BE1201/pKC283.
  • the single colony obtained was inocu­lated into 10 ml of LB medium containing 50 ⁇ g/ml ampicillin and incubated overnight at 32°C with vigorous shaking.
  • the 10 ml overnight culture was inoculated into 500 ml LB medium containing 50 ⁇ g/ml ampicillin and incubated at 32°C with vigorous shaking until the culture reached stationary phase.
  • the cells were harvested by centrifugation at 4000 g for 10 minutes at 4°C, and the supernatant was discarded.
  • the cell pellet was washed in 100 ml of ice-cold STE buffer (0.1 M NaCl; 10 mM Tris-HC1, pH 7.8; and 1 mM EDTA). After washing, the cell pellet was resuspended in 10 ml of Solution 1 (50 mM glucose; 25 mM Tris-HC1, pH 8.0; and 10 mM EDTA) containing 5 mg/ml lysozyme and left at room temperature for 10 minutes. Twenty ml of Solution 2 (0.2 N NaOH and 1% SDS) were then added to the lysozyme-treated cells, and the solution was gently mixed by inversion. The mixture was incubated on ice for 10 minutes.
  • the lysed cell mixture was centrifuged in a Beckman SW27 (or its equivalent) at 20,000 rpm for 20 minutes at 4°C.
  • the cell DNA and debris formed a pellet on the bottom of the tube.
  • About 36 ml of supernatant were recovered, and 0.6 volumes of isopropanol were added, mixed, and the resulting solution left at room temperature for 15 minutes.
  • the plasmid DNA was col­lected by centrifugation at 12,000 g for 30 minutes at room temperature.
  • the supernatant was discarded, and the DNA pellet was washed with 70% ethanol at room temperature.
  • the ethanol wash was decanted, and the pellet was dried in a vacuum desiccator.
  • the pellet was then resuspended in 8 ml of TE buffer (10 mM Tris-HC1, pH 8.0, and 1 mM EDTA).
  • CsCl Eight grams were added to the DNA solution. About 0.8 ml of a 10 mg/ml solution of ethidium bromide in water were added for each 10 ml of CsCl-DNA solution. The final density of the solution was about 1.55 g/ml, and the ethidium bromide con­centraton was about 600 ⁇ g/ml.
  • the solution was transferred to a Beckman Type 50 centrifuge tube, filled to the top with paraffin oil, sealed, and centrifuged at 45,000 rpm for 24 hours at 20°C. After centrifugation, two bands of DNA were visible in ordinary light. After removing the cap from the tube, the lower DNA band was removed by using a syringe with a #21 hypodermic needle inserted through the side of the centrifuge tube.
  • the ethidium bromide was removed by several extractions with water-saturated 1-butanol.
  • the CsCl was removed by dialysis against TE buffer. After extractions with buffered phenol and then chloroform, the DNA was precipitated, washed with 70% ethanol, and dried.
  • About 1 mg of plasmid pKC283 was obtained and stored at 4°C in TE buffer at a concentration of about 1 ⁇ g/ ⁇ l.
  • a restriction site and function map of plasmid pKC283 is presented in Figure 1 of the accompanying drawings.
  • Restriction enzyme reactions described herein were routinely terminated by phenol and then chloroform extractions, which were followed by precipitation of the DNA, an ethanol wash, and resus­pension of the DNA in TE buffer. After terminating the Pvu II digestion as described above, the Pvu II-digested plasmid pKC283 DNA was precipitated and then resuspended in 5 ⁇ l of TE buffer.
  • kinased Xho I linkers were added to the 5 ⁇ l of Pvu II-digested plasmid pKC283 DNA, and then 2.5 ⁇ l of 10 X ligase buffer (300 mM Tris-HC1, pH 7.6; 100 mM MgCl2; and 50 mM DTT), 2.5 ⁇ l of 1 mg/ml BSA, 7 ⁇ l of 5 mM ATP, 2.5 ⁇ l (about 2.5 units as defined by P-L Biochemicals) of T4 DNA ligase, 2.5 ⁇ l of 10 mM spermidine, and 3 ⁇ l of water were added to the DNA. The resulting ligation reaction was incubated at 4°C overnight.
  • the reaction mixture was adjusted to have the composition of high-salt buffer (0.1 M NaCl; 0.05 M Tris-HC1, pH 7.5; 10.0 mM MgCl2; and 1 mM DTT). About 10 ⁇ l (100 units) of restriction enzyme Xho I were added to the mixture, and the resulting reaction was incubated at 37°C for 2 hours.
  • high-salt buffer 0.1 M NaCl; 0.05 M Tris-HC1, pH 7.5; 10.0 mM MgCl2; and 1 mM DTT.
  • E. coli K12 MO( ⁇ +) can be obtained from the Northern Regional Research Laboratories in lyophylized form under the accession number NRRL B-15993.
  • E. coli K12 MO( ⁇ +) comprises the wild-type lambda pL cI repressor gene, so that transcription from the hybrid pL- lpp promoter of the present invention does not occur in E. coli K12 MO( ⁇ +) cells.
  • the lyophils are reconstituted, single colonies of MO( ⁇ +) are isolated, and a 10 ml overnight culture of the MO( ⁇ +)cells is prepared in substantial accordance with the procedure of Example 1, except that the temperature of incubation is 37°C and no ampicillin is used in the growth media.
  • Fifty ⁇ l of the overnight culture were used to inoculate 5 ml of LB media which also contained 10 mM MgSO4 and 10 mM MgCl2.
  • the culture was incubated at 37°C overnight with vigorous shaking.
  • the culture was diluted to 200 ml with LB media containing 10 mM MgSO4 and 10 mM MgCl2.
  • the diluted culture was incubated at 37°C with vigorous shaking until the absorbance at 550 nm (A550) was about 0.5, which indicated a cell density of about 1 x 108 cells/ml.
  • the culture was cooled for ten minutes in an ice-water bath, and the cells were then collected by centrifugation at 4000 g for 10 minutes at 4°C.
  • the cell pellet was resuspended in 100 ml of cold 10 mM MgSO4 and then immediately re-pelleted by centrifu­gation.
  • the cell pellet was resuspended in 100 ml of 30 mM CaCl2 and incubated on ice for 20 minutes.
  • the cells were again collected by centri­fugation and resuspended in 10 ml of 30 mM CaCl2. A one-half ml aliquot of the cells was added to the ligated DNA prepared in Example 2; the DNA had been made 30 mM in CaCl2.
  • the cell-DNA mixture was incubated on ice for one hour, heat-shocked at 42°C for 90 seconds, and then chilled on ice for about two minutes.
  • the cell-DNA mixture was diluted into 10 ml of LB media in 125 ml flasks and incubated at 37°C for one hour. One hundred ⁇ l aliquots were plated on LB-agar plates containing ampicillin and incubated at 37°C until colonies appeared.
  • Plasmid DNA isolation was performed on a smaller scale in accordance with the procedure of Example 1, but the CsC1 gradient step was omitted until the desired E. coli K12 MO( ⁇ +)/pKC283PX transformants were identified.
  • a restriction site and function map of plasmid pKC283PX is presented in Figure 1 of the accompanying drawings.
  • a DNA linker with single-stranded DNA ends characteristic of Bgl II and Xho I restriction enzyme cleavage was synthesized and kinased.
  • the linker was kinased in substantial accordance with the procedure of Example 2.
  • the DNA linker had the following structure:
  • the linker depicted above was synthesized from single-­stranded deoxyoligonucleotides by procedures well known in the art.
  • the single-stranded deoxyoligonucleotides can be synthesized with commercially available instru­ments, such as the 380A DNA Synthesizer marketed by Applied Biosystems (850 Lincoln Centre Drive, Foster City, CA 94404), which utilizes phosphoramidite chem­istry.
  • the linker and Bgl II- Xho I-digested plasmid pKC283PX were ligated in substantial accordance with the procedure of Example 2.
  • the ligated DNA constituted the desired plasmid pKC283-L.
  • a restriction site and function map of plasmid pKC283-L is presented in Figure 1 of the accompanying drawings.
  • the plasmid pKC283-L DNA was used to transform E. coli K12 MO( ⁇ +) and the resulting E. coli K12 MO( ⁇ +)/pKC283-L transformants were identified in substantial accordance with the procedure of Example 3.
  • plasmid pKC283-L DNA prepared in substantial accordance with the procedures of Example 1, were dissolved in 20 ⁇ l 10X high-salt buffer, 20 ⁇ l 1 mg/ml BSA, 5 ⁇ l ( ⁇ 50 units) restriction enzyme Xho I, and 155 ⁇ l of H2O, and the resulting reaction was incubated at 37°C for two hours.
  • the Xho I-digested plasmid pKC283-L DNA was then precipitated from the reaction mixture by the addition of three volumes of 95% ethanol and one-tenth volume of 3 M sodium acetate, incubation in a dry ice-ethanol bath for five minutes, and centrifugation.
  • the resulting DNA pellet was washed with 70% ethanol, dried, and resuspended in 2 ⁇ l 10X nick-translation buffer (0.5 M Tris-HC1, pH 7.2; 0.1 M MgSO4; and 1 mM DTT), 1 ⁇ l of a solution 2 mM in each of the deoxynucleotide triphosphates, 15 ⁇ l of H2O, 1 ⁇ l ( ⁇ 6 units as defined by P-L Biochemicals) of Klenow, which is the large fragment of E. coli DNA polymerase I, and 1 ⁇ l of 1 mg/ml BSA.
  • the resulting reaction was incubated at 25°C for 30 minutes; the reaction was stopped by incubating the solution at 70°C for five minutes.
  • Bam HI linkers (5′-CGGGATCCCG-3′) were kinased and ligated to the Xho I-digested, Klenow-treated plasmid pKC283-L DNA in substantial accordance with the pro­cedure of Example 2. After the ligation reaction, the DNA was digested with about 100 units of Bam HI for about 2 hours at 37°C in high-salt buffer. After the Bam HI digestion, the DNA was prepared for ligation in substantial accordance with the procedure of Example 2.
  • the ⁇ 5.9 kb Bam HI restriction fragment was circularized by ligation and transformed into E. coli K12 MO( ⁇ +) in substantial accordance with the procedures of Examples 2 and 3.
  • the E. coli K12 MO( ⁇ +)/pKC283-LB transformants were identified, and then plasmid pKC283-LB DNA was prepared in substantial accordance with the procedure of Example 1.
  • a restriction site and function map of plasmid pKC283-LB is presented in Figure 1 of the accompanying drawings.
  • plasmid pKC283PX About 10 ⁇ g of plasmid pKC283PX were digested with restriction enzyme Sal I in high-salt buffer, treated with Klenow, and ligated to Eco RI linkers (5′-GAGGAATTCCTC-3′) in substantial accordance with the procedure of Example 5, with the exception of the starting plasmid, restriction enzymes, and linkers used.
  • Eco RI which results in the excision of ⁇ 2.1 kb of DNA
  • the ⁇ 4.0 kb Eco RI restriction fragment was circularized by ligation to yield plasmid pKC283PRS.
  • the ligated DNA was used to transform E. coli K12 MO( ⁇ +) in sub­stantial accordance with the procedure of Example 3. After the E.
  • Plasmid pKC283PRS DNA was prepared in substantial accordance with the procedure of Example 1.
  • a restriction site and function map of plasmid pKC283PRS is presented in Figure 1 of the accompanying drawings.
  • plasmid pKC283PRS About 10 ⁇ g of plasmid pKC283PRS were digested in 200 ⁇ l of high-salt buffer with about 50 units each of restriction enzymes Pst I and Sph I. After incubating the reaction at 37°C for about 2 hours, the reaction mixture was electrophoresed on a 0.6% low-gelling-­temperature agarose (FMC Corporation, Marine Colloids Division, Rockland, Maine 04841) gel for 2-3 hours at ⁇ 130 V and ⁇ 75 mA in Tris-Acetate buffer.
  • FMC Corporation Low-gelling-­temperature agarose
  • the gel was stained in a dilute solution of ethidium bromide, and the band of DNA constituting the ⁇ 0.85 kb Pst I- Sph I restriction fragment, which was visualized with long-wave UV light, was cut from the gel in a small segment.
  • the volume of the segment was determined by weight and density of the segment, and an equal volume of 10 mM Tris-HCl, pH 7.6, was added to the tube containing the segment. The segment was then melted by incubation at 72°C. About 1 ug of the ⁇ 0.85 kb Pst I- Sph I restriction fragment of plasmid pKC283PRS was obtained in a volume of about 100 ⁇ l.
  • plasmid pKC283-LB was digested with restriction enzymes Pst I and Sph I, and the resulting ⁇ 3.0 kb restriction fragment was isolated by agarose gel electrophoresis and prepared for ligation.
  • Plasmid pL32 was transformed into E. coli K12 MO( ⁇ +) cells in substantial accordance with the procedure of Example 3. Plasmid pL32 DNA was prepared from the E.
  • coli K12 MO( ⁇ +)/pL32 transformants in substantial accordance with the pro­cedure of Example 1.
  • Analysis of the plasmid pL32 DNA demonstrated that more than one Eco RI linker attached to the Klenow-treated, Sal I ends of plasmid pKC283PX.
  • the presence of more than one Eco RI linker does not affect the utility of plasmid pL32 or derivatives of plasmid pL32 and can be detected by the presence of an Xho I restriction site, which is generated whenever two of the Eco RI linkers are ligated together.
  • plasmid pL32 may be constructed by carrying out the Sal I- Eco RI excision and ligation of the first paragraph of this Example upon plasmid pKC283-LB.
  • E. coli K12 RV308/pNM789 can be obtained from the Northern Regional Research Laboratories in lyophilized form under the accession number NRRL B-18216.
  • a re­striction site and function map of pNM789 is presented in Figure 2 of the accompanying drawings.
  • Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except that the tem­perature of incubation is 37°C.
  • Ten micrograms of pNM789 are suspended in 200 ⁇ l Pvu II buffer (50 mM Tris-HCl (pH 7.5), 60 mM NaCl and 6mM MgCl2). One unit of Pvu II is added and the reaction mix is incubated for 5 minutes at 37°C.
  • the enzyme is inactivated by heating 10 minutes at 65°C. 30 ⁇ l of 10X Bam HI buffer (200 mM Tris-HCl (pH 8.0), 1M NaCl and 70 mM MgCl2), 70 ⁇ l H2O and 10 units of Bam HI are next added and the reaction is incubated for 1 hour at 37°C. This is followed by the addition of 5 units of alkaline phosphatase and incu­bation for 1 hour at 65°C. The DNA fragments are separated on a 1 percent agarose gel, and a DNA fragment ( Figure 3) the size of a single cut fragment is purified.
  • a DNA linker with a blunt end and a Bam HI end is synthesized in substantial accordance with the teaching of Example 4.
  • This linker (shown in Figure 3) has the following structure: The linker is kinased and ligated into the Bam HI- Pvu II digested plasmid pNM789 in substantial accordance with the teaching of Example 2. This ligation mixture is used to transform E. coli K12 RV308 cells and plasmid isolation is performed upon these transformants in substantial accordance with the teaching of Example 3. Several plasmids are selected which contain the appro­priate size Pvu II fragment (494bp) and Xba I- Bam HI fragment (628bp).
  • plasmid 120 This intermediate plasmid is designated plasmid 120.
  • a schematic outline of this procedure and a restriction site and function map of plasmid 120 is presented in Figure 3 of the accompanying drawings.
  • EK-BGH-encoding DNA about 10 ⁇ g of plasmid 120 were digested in 200 ⁇ l of high-salt buffer containing about 50 units each of restriction enzymes Xba I and Bam HI. The digestion products were separated by agarose gel electrophoresis, and the ⁇ 0.6 kb Xba I- Bam HI restriction fragment which encodes EK-BGH was isolated and prepared for ligation in substantial accordance with the procedure of Example 6.
  • Plasmid pL32 was also digested with restric­tion enzymes Xba I and Bam HI, and the ⁇ 3.9 kb restriction fragment was isolated and prepared for ligation.
  • the ⁇ 3.9 kb Xba I- Bam HI restriction fragment of plasmid pL32 was ligated to the ⁇ 0.6 kb Xba I- Bam HI restriction fragment of plasmid 120 in substantial accordance with the procedure of Example 2 to yield plasmid pL47.
  • a restriction site and function map of plasmid pL47 is presented in Figure 4 of the accompanying drawings.
  • Plasmid pL47 was transformed into E. coli K12 MO( ⁇ +) in substantial accordance with the procedure of Example 3, and the E. coli K12 MO( ⁇ +)/pL47 transformants were identified.
  • Plasmid pL47 DNA was prepared from the transformants in substantial accordance with the pro­cedures of Example 1.
  • Plasmid pPR12 comprises the temperature-­sensitive pL repressor gene cI857 and the plasmid pBR322 tetracycline resistance-conferring gene. Plasmid pPR12 is disclosed and claimed in U.S. Patent #4,436,815, issued 13 March 1984. A restriction site and function map of plasmid pPR12 is presented in Figure 4 of the accompanying drawings.
  • plasmid pPR12 About 10 ⁇ g of plasmid pPR12 were digested with about 50 units of restriction enzyme Eco RI in 200 ⁇ l of high-salt buffer at 37°C for two hours.
  • the Eco RI-digested plasmid pPR12 DNA was precipitated and treated with Klenow in substantial accordance with the procedure of Example 5.
  • Klenow reaction the Eco RI-digested, Klenow-treated plasmid pPR12 DNA was recircularized by ligation in substantial accordance with the procedure of Example 2.
  • the ligated DNA which constituted the desired plasmid pPR12 ⁇ R1, was used to transform E.
  • E. coli K12 RV308 in substantial accordance with the procedure of Example 3, except that selection was based on tetracycline (5 ug/ml) resistance, not ampicillin resistance.
  • E. coli K12 RV308 is available from the NRRL under the accession number NRRL B-15624. After the E. coli K12 RV308/pPR12 ⁇ R1 transformants were identified, plasmid pPR12 ⁇ R1 DNA was prepared from the transformants in substantial accordance with the pro­cedure of Example 1.
  • plasmid pPR12 ⁇ R1 About 10 ⁇ g of plasmid pPR12 ⁇ R1 were digested with about 50 units of restriction enzyme Ava I in 200 ⁇ l of medium-salt buffer at 37°C for 2 hours.
  • the Ava I-­digested plasmid pPR12 ⁇ R1 DNA was precipitated and treated with Klenow in substantial accordance with the procedure of Example 5.
  • Klenow reaction the Ava I-digested, Klenow-treated plasmid pPR12 ⁇ R1 DNA was ligated to Eco R1 linkers (5′-GAGGAATTCCTC-3′) in sub­stantial accordance with the procedure of Example 2.
  • the DNA was precipitated and then resuspended in about 200 ⁇ l of high-salt buffer containing about 50 units of restriction enzyme Eco R1.
  • the resulting reaction was incubated at 37°C for about 2 hours.
  • the reaction mixture was loaded onto an agarose gel, and the ⁇ 5.1 kb Eco R1 restriction fragment was purified in substantial accordance with the procedure of Example 6.
  • the ⁇ 5.1 kb Eco R1 restriction fragment was recircularized by ligation in substantial accordance with the procedure of Example 2.
  • the ligated DNA constituted the desired plasmid PPR12AR1.
  • the plasmid pPR12AR1 DNA was transformed into E.
  • plasmid pPR12AR1 DNA was prepared in sub­stantial accordance with the procedure of Example 1.
  • a restriction site and function map of plasmid pPR12AR1 is presented in Figure 4 of the accompanying drawings.
  • plasmid pPR12AR1 DNA About 10 ⁇ g of plasmid pPR12AR1 DNA were suspended in about 200 ml of high-salt buffer containing about 50 units each of restriction enzymes Pst I and Eco RI, and the digestion reaction was incubated at 37°C for about 2 hours. The reaction mixture was then loaded onto an agarose gel, and the ⁇ 2.9 kb Pst I- Eco RI restric­tion fragment of plasmid pPR12AR1 was isolated and prepared for ligation in substantial accordance with the procedure of Example 6.
  • plasmid pL47 About 10 ug of plasmid pL47 were digested with restriction enzymes Pst I and Bam HI in 200 ul of high-salt buffer at 37°C for two hours.
  • the Pst I- Bam HI-digested DNA was loaded onto an agarose gel, and the ⁇ 2.7 kb Pst I- Bam HI restriction fragment that comprised the origin of replication and a portion of the ampicillin resistance-conferring gene was isolated and prepared for ligation in substantial accordance with the procedure of Example 6.
  • BK virus is obtained from the American Type Culture Collection under the accession number ATCC VR-837. The virus is delivered in freeze-dried form and resuspended in Hank's balanced salts (Gibco, 3175 Staley Road, Grand Island, NY 14072) to a titer of about 105 plaque-forming units (pfu)/ml.
  • the host of choice for the preparation of BK virus DNA is primary human embryonic kidney (PHEK) cells, which can be obtained from Flow Laboratories, Inc., 7655 Old Springhouse Road, McLean, VA 22101; under catalogue number 0-100 or from M.A. Bioproducts under catalogue number 70-151.
  • PHEK primary human embryonic kidney
  • the virus is released from the cells by three freeze-thaw cycles, and the cellular debris is removed by centrifugation at 5000Xg.
  • the virus in 1 liter of supernatant fluid is precipitated and collected by the addition of 100 g of PEG-6000, incubation of the solu­tion for 24 hours at 4°C, and centrifugation at 5000Xg for 20 minutes.
  • the virus suspension is layered onto a 15 ml solution of saturated KBr in a tube, which is centrifuged at 75,000Xg for 3 hours.
  • SDS Sodium dodecyl sulfate
  • pronase is added to a concen­tration of 100 ⁇ g/ml, and the solution is incubated at 37°C for 2 hours.
  • Cesium chloride is then added to the solution to a density of 1.56 g/ml, and ethidium bromide is added to the solution to a final concentra­tion of 100 ⁇ g/ml.
  • the solution is centrifuged in a Sorvall (DuPont Inst. Products, Biomedical Division, Newton, CT 06470) 865 rotor or similar vertical rotor at 260,000Xg for 24 hours.
  • the solution of BK virus DNA is then dialyzed against TE buffer until the 260 nm/280 nm absorbance ratio of the DNA is between 1.75 and 1.90.
  • the DNA is precipitated by adjusting the NaCl concentration to 0.15 M, adding two volumes of ethanol, incubating the solution at -70°C for at least 2 hours, and centrifuging the solution at 12,000Xg for 10 minutes.
  • the resulting pellet of BK virus DNA is suspended in TE buffer at a concentration of 1 mg/ml.
  • a restriction site and function map of the BK virus is presented in Figure 5 of the accompanying drawings.
  • E. coli K12 HB101/pdBPV-MMTneo cells are obtained in lyophil form from the American Type Culture Collection under the accession number ATCC 37224.
  • the lyophilized cells are plated on L-agar plates containing 100 ⁇ g/ml ampicillin and incubated at 37°C to obtain single colony isolates.
  • L broth (10 g tryptone, 10 g NaCl, and 5 g yeast extract per liter) containing 50 ⁇ g/ml ampicillin was inoculated with a colony of E. coli K12 HB101/pdBPV-MMTneo and incubated in an air­shaker at 37°C until the O.D.590 was ⁇ 1 absorbance unit, at which time 150 mg of chloramphenicol were added to the culture. The incubation was continued for about 16 hours; the chloramphenicol addition inhibits protein synthesis, and thus inhibits further cell division, but allows plasmid replication to continue.
  • Plasmid DNA was then isolated from this culture in substantial accordance with the teaching of Example 1 and the ⁇ 1 mg of plasmid pdBPV-MMTneo DNA obtained by this procedure was suspended in 1 ml of TE buffer and stored at -20°C.
  • Plasmid pBKneo1 contains an ⁇ 2.1 kb Sal I-­ Hin dIII restriction fragment.
  • E. coli K12 HB101 cells are available in lyophilized form from the Northern Regional Research Laboratory under the accession number NRRL B-15626.
  • E. coli K12 HB101 cells were cultured, made competent for transformation, and transformed with the ligated DNA prepared above in substantial accordance with the procedure of Example 3. The transformed cells were plated on L-agar plates containing 100 ⁇ g/ml ampicillin.
  • E. coli K12 HB101/pBKneo1 and E. coli K12/pBKneo2 transformants were identified by their ampicillin-­resistant phenotype and by restriction enzyme analysis of their plasmid DNA.
  • a restriction site and function map of plasmid pBKneo1 is presented in Figure 5 of the accompanying drawings.
  • the virion DNA of adenovirus 2 is a double-stranded linear molecule about 35.94 kb in size.
  • the Ad2 late promoter can be isolated on an ⁇ 0.316 kb Acc I- Pvu II restriction fragment of the Ad2 genome; this ⁇ 0.32 kb restriction fragment corresponds to the sequence between nucleotide positions 5755 and 6071 of the Ad2 genome.
  • Ad2 DNA is first digested with restriction enzyme Bal I, and the ⁇ 2.4 kb Bal I restric­tion fragment that comprises the entire sequence of the ⁇ 0.32 kb Acc I- Pvu II restriction fragment is isolated. Then, the ⁇ 2.4 kb Bal I restriction fragment is digested with Acc I and Pvu II to obtain the desired fragment.
  • Ad2 DNA available from BRL or ATCC VR-2
  • 10 ⁇ l ( ⁇ 20 units) of restriction enzyme Bal I are added to the solution of Ad2 DNA, and the resulting reaction is incubated at 37°C for 4 hours.
  • the Bal I-digested DNA is loaded onto an agarose gel and electrophoresed until the restriction fragments are well separated. Visualization of the electrophoresed DNA is accomplished by staining the gel in a dilute solution (0.5 ⁇ g/ml) of ethidium bromide and exposing the stained gel to long-wave ultraviolet (UV) light.
  • a dilute solution 0.5 ⁇ g/ml
  • UV long-wave ultraviolet
  • One method to isolate DNA from agarose is as follows. A small slit is made in the gel in front of the desired fragment, and a small piece of NA-45 DEAE membrane (Schleicher and Schuell, Keene, NH 03431) is placed in each slit. Upon further electrophoresis, the DNA non-covalently binds to the DEAE membrane.
  • the volume of the high salt-DNA solution is adjusted so that the NaCl concentration is 0.25 M, and then three volumes of cold, absolute ethanol are added to the solution.
  • the resulting solution is mixed and placed at -70°C for 10-20 minutes.
  • the solution is then centrifuged at 15,000 rpm for 15 minutes. After another precipitation to remove residual salt, the DNA pellet is rinsed with ethanol, dried, resuspended in 20 ⁇ l of TE buffer, and constitutes about 3 ⁇ g of the desired restriction fragment of Ad2.
  • the purified fragment obtained is dissolved in 10 ⁇ l of TE buffer.
  • the Acc I- Pvu II-digested, ⁇ 2.4 kb Bal I restric­tion fragment of Ad2 is loaded onto an ⁇ 6% polyacryl­amide gel and electrophoresed until the ⁇ 0.32 kb Acc I- Pvu II restriction fragment that comprises the Ad2 late promoter is separated from the other digestion products.
  • the gel is stained with ethidium bromide and viewed using UV light, and the segment of gel containing the ⁇ 0.32 kb Acc I- Pvu II restriction fragment is cut from the gel, crushed, and soaked overnight at room temperature in ⁇ 250 ⁇ l of extraction buffer (500 mM NH4OAc; 10 mM MgOAc; 1 mM EDTA; and 0.1% SDS).
  • the mixture is centrifuged, and the pellet is discarded.
  • the DNA in the supernatant is precipitated with ethanol; about 2 ⁇ g of tRNA are added to ensure complete precipitation of the desired frag­ment.
  • About 0.2 ⁇ g of the ⁇ 0.32 kb Acc I- Pvu II restric­tion fragment are obtained and suspended in 7 ⁇ l of H2O.
  • Bcl I linkers (5′-CTGATCAG-3′, available from New England Biolabs), which had been kinased in substantial accordance with the procedure described in Example 2 was added to the solution of the ⁇ 0.32 kb Acc I- Pvu II restriction frag­ment, and then, 1 ⁇ l ( ⁇ 1000 units) of T4 DNA ligase and 1 ⁇ l of 10X ligase buffer were added to the solution of DNA, and the resulting reaction was incubated at 16°C overnight.
  • the Bcl I linkers could only ligate to the Pvu II end of the Acc I- Pvu II restriction fragment.
  • E. coli K12 HB101/pSV2cat cells are obtained in lyophilized form from the ATCC under the accession number ATCC 37155, and plasmid pSV2cat DNA was isolated from the cells in substantial accordance with the pro­cedure of Example 1.
  • About 1 mg of plasmid pSV2cat DNA is obtained and dissolved in 1 ml of TE buffer.
  • About 3 ⁇ g (3 ⁇ l) of the plasmid pSV2cat DNA were added to 2 ⁇ l of 10X Acc I buffer and 16 ⁇ l of H2O, and then, 3 ⁇ l (about 9 units) of restriction enzyme Acc I were added to the solution of pSV2cat DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • Acc I- Stu I-digested plasmid pSV2cat DNA About 4 ⁇ l of the Acc I- Stu I-digested plasmid pSV2cat DNA were mixed with about 7 ⁇ l of the ⁇ 0.32 kb Acc I- Pvu II (with Bcl I linkers attached) restriction fragment of Ad2, and after the addition of 3 ⁇ l of 10X ligase buffer, 15 ⁇ l of H2O, and 2 ⁇ l (about 1000 units) of T4 DNA ligase, the ligation reaction was incubated at 16°C overnight.
  • the ligated DNA consti­tuted the desired plasmid pLPcat, a plasmid that com­prises the Ad2 late promoter positioned so as to drive transcription, and thus expression, of the chloram­phenicol acetyltransferase gene.
  • a restriction site and function map of plasmid pLPcat is presented in Figure 6 of the accompanying drawings.
  • the ligated DNA was used to transform E. coli K12 HB101 cells in substantial accordance with the pro­cedure of Example 3.
  • the transformed cells were plated on L agar containing 50 ⁇ g/ml ampicillin; restriction enzyme analysis of plasmid DNA was used to identify the E. coli K12 HB101/pLPcat transformants. Plasmid pLPcat DNA was isolated from the transformants for use in subsequent constructions in substantial accordance with the plasmid isolation procedure described in Example 1.
  • plasmid pLPcat DNA was dis­solved in 5 ⁇ l of 10X Acc I buffer and 40 ⁇ l of H2O.
  • About 5 ⁇ l ( ⁇ 25 units) of restriction enzyme Acc I were added to the solution of plasmid pLPcat DNA, and the resulting reaction was incubated at 37°C.
  • the Acc I-digested plasmid pLPcat DNA was precipitated with ethanol and resuspended in 5 ⁇ l of 10X Stu I buffer, 40 ⁇ l of H2O, and 5 ⁇ l (about 25 units) of restriction enzyme Stu I, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Acc I- Stu I-digested plasmid pLPcat DNA was precipitated with ethanol several times to purify the ⁇ 4.81 kb Acc I- Stu I restriction fragment that comprises the E. coli origin of replication and Ad2 late promoter away from the other digestion product, a restriction fragment about 16 bp in size. About 1 ⁇ g of the desired ⁇ 4.81 kb restriction fragment was obtained and dissolved in 20 ⁇ l of TE buffer.
  • E. coli K12 HB101 cells were used to transform E. coli K12 HB101 cells in substantial accordance with the procedure described in Example 3.
  • E. coli K12 HB101/pBLcat transformants were identified by restric­tion enzyme analysis of their plasmid DNA.
  • Plasmid pBLcat DNA was prepared for use in subsequent construc­tions in substantial accordance with the procedure of Example 1.
  • Plasmid pHC7 comprises a DNA sequence that encodes human protein C.
  • One liter of L-broth con­taining 15 ⁇ g/ml tetracycline was inoculated with a culture of E. coli K12 RR1/pHC7 (NRRL B-15926), and plasmid pHC7 DNA was isolated and purified in substan­tial accordance with the procedure of Example 1.
  • a restriction site and function map of plasmid pHC7 is presented in Figure 7 of the accompanying drawings. About 1 mg of plasmid pHC7 DNA was obtained by this procedure, suspended in 1 ml of TE buffer, and stored at -20°C.
  • the Ban I-digested plasmid pHC7 DNA was then electrophoresed on a 3.5% polyacrylamide gel (29:1, acrylamide:bis­acrylamide), until the ⁇ 1.25 kb Ban I restriction frag­ment was separated from the other digestion products.
  • the region of the gel containing the ⁇ 1.25 kb Ban I restriction fragment was cut from the gel, placed in a test tube, and broken into small fragments.
  • One ml of extraction buffer 500 mM NH4OAc, 10 mM MgOAc, 1 mM EDTA, 1% SDS, and 10 mg/ml tRNA was added to the tube containing the fragments, and the tube was placed at 37°C overnight. Centrifugation was used to pellet the debris; and the supernatant was transferred to a new tube.
  • the debris was washed once with 200 ⁇ l of ex­traction buffer; the wash supernatant was combined with the first supernatant from the overnight extraction.
  • Ban I restriction fragment Approximately 8 ⁇ g of the ⁇ 1.25 kb Ban I restriction fragment were obtained by this procedure. The purified fragment was suspended in 10 ⁇ l of TE buffer and stored at -20°C. The Ban I restriction fragment had to be modified by the addition of a linker to construct plasmid pSV2-HPC8.
  • reaction buffer which contained 15 units ( ⁇ 0.5 ⁇ l) T4 polynucleotide kinase, 2 ⁇ l 10X ligase buffer, 10 ⁇ l of 500 ⁇ M ATP, and 7.5 ⁇ l of H2O.
  • the kinase reaction was incubated at 37°C for 30 minutes, and the reaction was terminated by incubation at 100°C for 10 minutes.
  • reaction was chilled on ice, 2 ⁇ l of 0.2 M dithiothreitol, 2.5 ⁇ l of 5 mM ATP, and 15 units of T4 polynucleotide kinase were added to the reaction mixture and mixed, and the reaction mixture was incubated another 30 minutes at 37°C. The reaction was stopped by another 10 minute incubation at 100°C and then chilled on ice.
  • the two single strands of the DNA linker were mixed together after the kinase reaction.
  • the kinase reaction mixture was incubated at 100°C for 10 minutes in a water bath containing ⁇ 150 ml of water. After this incubation, the water bath was shut off and allowed to cool to room temperature, a process taking about 3 hours. The water bath, still containing the tube of kinased DNA, was then incubated at 4°C overnight. This process annealed the single strands.
  • the linker con­structed had the following structure: The linker was stored at -20°C until use.
  • the ⁇ 8 ⁇ g of ⁇ 1.25 kb Ban I fragment were added to and mixed with the ⁇ 50 ⁇ l of linker ( ⁇ 500 picomoles), 1 ⁇ l of T4 DNA ligase ( ⁇ 500 units), 10 ⁇ l of 10X ligase buffer, and 29 ⁇ l of H2O, and the resulting ligation reaction was incubated at 4°C overnight. The ligation reaction was stopped by a 10 minute incubation at 65°C. The DNA was pelleted by adding NaOAc to a final concen­tration of 0.3 M, adding 2 volumes of ethanol, chilling in a dry ice-ethanol bath, and then centrifuging the solution.
  • 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Apa I and 85 ⁇ l of H
  • the reaction mixture was loaded onto a 3.5% polyacrylamide gel, and the desired ⁇ 1.23 kb Hin dIII-­ Apa I restriction fragment was isolated in substantial accordance with the procedure described in Example 12A. Approximately 5 ⁇ g of the desired fragment were obtained, suspended in 10 ⁇ l of TE buffer, and stored at -20°C.
  • the Pst I-­digested plasmid pHC7 DNA was then electrophoresed on a 3.5% polyacrylamide gel, and the desired ⁇ 0.88 kb fragment was purified in substantial accordance with the procedure described above. Approximately 5 ⁇ g of the desired fragment were obtained, suspended in 10 ⁇ l of TE buffer, and stored at -20°C.
  • the ⁇ 5 ⁇ g of ⁇ 0.88 kb Pst I fragment were added to and mixed with ⁇ 50 ⁇ l of the following linker, which was constructed on an automated DNA synthesizer: About 1 ⁇ l of T4 DNA ligase ( ⁇ 10 units), 10 ⁇ l 10X ligase buffer, and 29 ⁇ l H2O were added to the mixture of DNA, and the resulting ligation reaction was incu­bated at 4°C overnight.
  • the ligation reaction was stopped by a 10 minute incubation at 65°C. After precipitation of the ligated DNA, the DNA pellet was dissolved in 10 ⁇ l of 10X Apa I reaction buffer, 5 ⁇ l ( ⁇ 50 units) of restric­tion enzyme Apa I, and 85 ⁇ l of H2O, and the reaction was placed at 37° for two hours. The reaction was then stopped and the DNA pelleted once again.
  • plasmid pSV2gpt DNA ATCC 37145
  • 10X Hin dIII reaction buffer 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Hin dIII, and 85 ⁇ l of H2O
  • the reaction mixture was then made 0.25 M in NaOAc, and after the addition of two volumes of ethanol and incubation in a dry ice-ethanol bath, the DNA was pelleted by centrifugation.
  • the DNA pellet was dissolved in 10 ⁇ l of 10X Bgl II buffer, 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Bgl II, and 85 ⁇ l of H2O, and the reaction was placed at 37°C for two hours. After the Bgl II digestion, the reaction mixture was loaded onto a 1% agarose gel, and the fragments were separated by electrophoresis. The gel was stained with ethidium bromide and viewed under ultraviolet light, and the band containing the desired ⁇ 5.1 kb Hin dIII- Bgl II fragment was cut from the gel and placed in dialysis tubing, and electrophoresis was continued until the DNA was out of the agarose.
  • the buffer containing the DNA from the dialysis tubing was extracted with phenol and CHCl3, and then, the DNA was precipitated.
  • the pellet was resus­pended in 10 ⁇ l of TE buffer and constituted ⁇ 5 ⁇ g of the desired ⁇ 5.1 kb Hin dIII- Bgl II restriction fragment of plasmid pSV2gpt.
  • E. coli K12 RR1 (NRRL B-15210) cells were made competent for transformation in substantial accordance with the procedure described in Example 3.
  • the ligated DNA prepared above was used to transform the cells, and aliquots of the transformation mix were plated on L-agar plates containing 100 ⁇ g/ml ampicillin. The plates were then incubated at 37°C.
  • E. coli K12 RR1/pSV2-HPC8 transformants were verified by restriction enzyme analysis of their plasmid DNA.
  • the resulting Sal I reaction mixture was incu­bated for 2 hours at 37°C.
  • the Hin dIII- Sal I-digested plasmid pSV2-HPC8 was loaded onto a 3.5% polyacrylamide gel and electrophoresed until the desired ⁇ 0.29 kb Hin dIII- Sal I restriction fragment was separated from the other reaction products.
  • the desired fragment was isolated from the gel; about 2 ⁇ g of the fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • plasmid pSV2-HPC8 Fifty ⁇ g of plasmid pSV2-HPC8 were dissolved in 10 ⁇ l of 10X Bgl II reaction buffer, 5 ⁇ l (50 units) of restriction enzyme Bgl II, and 85 ⁇ l of H2O, and the reaction was incubated at 37°C for two hours. After the Bgl II digestion, the DNA was precipitated, and the DNA pellet was dissolved in 10 ⁇ l of 10X Sal I reaction buffer, 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Sal I, and 85 ⁇ l of H2O. The resulting Sal I reaction mixture was incubated for 2 hours at 37°C.
  • the Sal I- Bgl II-digested plasmid pSV2-HPC8 was loaded onto a 3.5% polyacrylamide gel and electrophoresed until the desired ⁇ 1.15 kb Sal I- Bgl II restriction fragment was separated from the other reaction products.
  • the ⁇ 1.15 kb Sal I- Bgl II restriction fragment was isolated from the gel; about 8 ⁇ g of fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • plasmid pSV2- ⁇ -globin DNA (NRRL B-15928) were dissolved in 10 ⁇ l of 10X Hin dIII reaction buffer, 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Hin dIII, and 85 ⁇ l of H2O, and the reaction was placed at 37°C for 2 hours. The reaction mixture was then made 0.25 M in NaOAc, and after the addition of two volumes of ethanol and incubation in a dry ice-ethanol bath, the DNA was pelleted by centrifugation.
  • Hin dIII-digested plasmid pSV2- ⁇ -globin was dissolved in 10 ⁇ l of 10X Bgl II buffer, 5 ⁇ l ( ⁇ 50 units) of restric­tion enzyme Bgl II, and 85 ⁇ l of H2O, and the reaction was placed at 37°C for two hours. After the Bgl II digestion, the reaction mixture was loaded onto a 1% agarose gel, and the fragments were separated by electrophoresis. The desired ⁇ 4.2 kb Hin dIII- Bgl II restriction fragment was isolated from the gel; about 5 ⁇ g of the desired fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • Example 13A Two ⁇ l of the ⁇ 0.29 kb Hin dIII- Sal I fragment of plasmid pSV2-HPC8, 2 ⁇ l of the ⁇ 1.15 kb Sal I- Bgl II fragment of plasmid pSV2-HPC8, and 2 ⁇ l of the ⁇ 4.2 kb Hin dIII- Bgl II fragment of plasmid pSV2- ⁇ -globin were mixed together and ligated in substantial accordance with the procedure of Example 13A.
  • the ligated DNA constituted the desired plasmid pL133.
  • a restriction site and function map of plasmid pL133 is presented in Figure 7 of the accompanying drawings.
  • the desired E. coli K12 RR1/pL133 transformants were constructed in substantial accordance with the teaching of Example 16A, with the exception that plasmid pL133, rather than plasmid pSV2-HPC8, was used as the transforming DNA.
  • plasmid pBLcat DNA About 20 ⁇ g of plasmid pBLcat DNA were dis­solved in 10 ⁇ l of 10X Hin dIII buffer and 80 ⁇ l of H2O. About 10 ⁇ l ( ⁇ 100 units) of restriction enzyme Hin dIII were added to the solution of plasmid pBLcat DNA, and the resulting reaction was incubated at 37°C for 2 hours. The Hin dIII-digested plasmid pBLcat DNA was loaded onto an agarose gel and electrophoresed until the ⁇ 0.87 kb Hin dIII restriction fragment that comprises the BK enhancer and Ad2 late promoter was separated from the other digestion products; then, the ⁇ 0.87 kb fragment was isolated and prepared for ligation in substantial accordance with the procedure of Example 12A. About 2 ⁇ g of the desired fragment were obtained and dissolved in 5 ⁇ l of TE buffer.
  • plasmid pL133 DNA was dis­solved in 2 ⁇ l of 10X Hin dIII buffer and 16 ⁇ l of H2O. About 1 ⁇ l ( ⁇ 10 units) of restriction enzyme Hin dIII was added to the solution of DNA, and the resulting reaction was incubated at 37°C for 2 hours. The DNA was then diluted to 100 ⁇ l with TE buffer and treated with calf-intestinal alkaline phosphatase in substantial accordance with the procedure in Example 7. The Hin dIII-digested plasmid pL133 DNA was extracted twice with phenol and once with chloroform, precipitated with ethanol, and resuspended in 10 ⁇ l of TE buffer.
  • the ligated DNA was used to transform E. coli K12 HB101 in substantial accordance with the procedure of Example 3.
  • the transformed cells were plated on L agar containing ampicillin, and the plasmid DNA of the ampicillin-resistant transformants was examined by restriction enzyme analysis to identify the E. coli K12 HB101/pLPC transformants.
  • the ⁇ 0.87 kb Hin dIII restriction fragment that encodes the BK enhancer and Ad2 late promoter could insert into Hin dIII-digested plasmid pL133 in one of two orientations, only the construction which contains an ⁇ l.0 kb Nde I- Stu I frag­ment yields pLPC.
  • E. coli K12 RR1/pSV2hyg cells are obtained from the Northern Regional Research Laboratory under the accession number NRRL B-18039. Plasmid pSV2hyg DNA is obtained from the cells in substantial accordance with the procedure of Example 1. A restriction site and function map of plasmid pSV2hyg is presented in Figure 8 of the accompanying drawings.
  • the Klenow-­treated, Bam HI-digested plasmid pSV2hyg DNA was extracted once with phenol and once with chloroform and then precipitated with ethanol. About 2 ⁇ g of the desired fragment were obtained and suspended in 5 ⁇ l of TE buffer.
  • the Nde I- Stu I-digested plasmid pLPC DNA was precipitated with ethanol, collected by centrifugation, and resuspended in 5 ⁇ l of 10X Klenow buffer and 40 ⁇ l of H2O. About 5 ⁇ l ( ⁇ 25 units) of Klenow enzyme were added to the solution of DNA, and the resulting reaction was incubated at 16°C for 30 minutes. After the Klenow reaction, the reaction mixture was loaded onto an agarose gel, and the ⁇ 5.82 kb Nde I- Stu I restriction fragment was isolated from the gel. About 5 ⁇ g of the desired fragment were obtained and suspended in 5 ⁇ l of TE buffer.
  • the ligated DNA constituted the desired plasmids pLPChyg1 and pLPChyg2, which differ only with respect to the orientation of the ⁇ 2.5 kb Klenow-treated, Bam HI restriction fragment of plasmid pSV2hyg.
  • a restriction site and function map of plasmid pLPChyg1 is presented in Figure 8 of the accompanying drawings.
  • the ligated DNA was used to transform E. coli K12 HB101 in substantial accordance with the procedure of Example 3.
  • the desired E. coli K12 HB101/pLPChyg1 and E. coli K12 HB101/pLPChyg2 trans­formants were plated on L agar containing ampicillin and identified by restriction enzyme analysis of their plasmid DNA.
  • Plasmid pTPA102 comprises the coding sequence of human tissue plasminogen activator (TPA). Plasmid pTPA102 can be isolated from E. coli K12 MM294/pTPA102, a strain available from the Northern Regional Research Laboratory under the accession number NRRL B-15834. A restriction site and function map of plasmid pTPA102 is presented in Figure 9 of the accompanying drawings. Plasmid pTPA102 DNA is isolated from E. coli K12 MM294/pTPA102 in substantial accordance with the pro­cedure of Example 1.
  • plasmid pTPA102 in about 50 ⁇ l of TE buffer
  • 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Tth 111I were added to the solution of DNA, and the resulting reaction was incubated at 65°C for 2 hours.
  • the reaction mixture was loaded onto an agarose gel, and the ⁇ 4.4 kb Tth 111I restriction fragment that com­prises the TPA coding sequence was isolated from the gel.
  • 10 ⁇ g of the desired ⁇ 4.4 kb Tth 111I restriction fragment were obtained and suspended in 10 ⁇ l of TE buffer.
  • kinased Bam HI linkers were added to the solution of ⁇ 4.4 kb Tth 111I restriction fragment, and after the addition of 2 ⁇ l of T4 DNA ligase ( ⁇ 1000 units) and 1 ⁇ l of T4 RNA ligase ( ⁇ 2 units), the ligation reaction was incubated overnight at 4°C. The ligated DNA was precipitated with ethanol and resuspended in 5 ⁇ l of 10X Hin dIII buffer and 40 ⁇ l of H2O. About 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Hin dIII were added to the solution of DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Hin dIII-digested DNA was precipitated with ethanol and resuspended in 10 ⁇ l of 10X Bam HI buffer and 90 ⁇ l of H2O. About 10 ⁇ l ( ⁇ 100 units) of restriction enzyme Bam HI were added to the solution of DNA, and the resulting reaction was incubated at 37°C for 2 hours. After the Bam HI digestion, the reaction mixture was loaded onto an agarose gel, and the ⁇ 2.0 kb Bam HI- Hin dIII restriction fragment was isolated from the gel. About 4 ⁇ g of the desired fragment were obtained and suspended in about 5 ⁇ l of TE buffer.
  • Plasmid pRC was constructed by inserting an ⁇ 288 bp Eco RI- Cla I restriction fragment that comprises the promoter and operator ( trp PO) sequences of the E. coli trp operon into Eco RI- Cla I-digested plasmid pKC7. Plasmid pKC7 can be obtained from the American Type Culture Collection in E. coli K12 N100/pKC7 under the accession number ATCC 37084.
  • the ⁇ 288 bp Eco RI- Cla I restriction fragment that comprises the trp PO can be isolated from plasmid pTPA102, which can be isolated from E. coli K12 MM294/pTPA102 (NRRL B-15834). Plasmid pKC7 and plasmid pTPA102 DNA can be obtained from the aforementioned cell lines in substantial accordance with the procedure of Example 1.
  • This ⁇ 0.29 kb Eco RI- Cla I restriction fragment of plasmid pTPA102 comprises the transcription activating sequence and most of the translation activating sequence of the E. coli trp gene and has the sequence depicted below:
  • the Cla I-digested plasmid pKC7 DNA was precipitated with ethanol and resuspended in 2 ⁇ l of 10X Eco RI buffer and 16 ⁇ l of H2O. About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Eco RI were added to the solution of Cla I-digested plasmid pKC7 DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Eco RI- Cla I-digested plasmid pKC7 DNA was extracted once with phenol and then twice with chloro­form. The DNA was then precipitated with ethanol and resuspended in 3 ⁇ l of 10X ligase buffer and 20 ⁇ l of H2O.
  • a restriction site and function map of plasmid pKC7 can be obtained from Maniatis et al ., Molecular Cloning (Cold Spring Harbor Laboratory, 1982), page 8.
  • plasmid pTPA102 in about 20 ⁇ l of TE buffer were added to 10 ⁇ l of 10X Cla I buffer and 60 ⁇ l of H2O.
  • About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Cla I were added to the solution of plasmid pTPA102 DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Cla I-digested plasmid pTPA102 DNA was precipitated with ethanol and resuspended in 10 ⁇ l of 10X Eco RI buffer and 80 ⁇ l of H2O.
  • About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Eco RI were added to the solution of Cla I-digested plasmid pTPA102 DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Eco RI- Cla I-digested plasmid pTPA102 DNA was extracted once with phenol, loaded onto a 7% poly­acrylamide gel, and electrophoresed until the ⁇ 288 bp Eco RI- Cla I restriction fragment that comprises the trp PO was separated from the other digestion products.
  • the ⁇ 288 bp Eco RI- Cla I restriction fragment was isolated from the gel; about 1 ⁇ g of the desired fragment was obtained, suspended in 5 ⁇ l of TE buffer, and added to the solution of Eco RI- Cla I-digested plasmid pKC7 DNA prepared as described above.
  • the ligated DNA constituted the desired plasmid pRC DNA.
  • the ligated DNA was used to transform E. coli K12 HB101 competent cells in substantial accordance with the procedure of Example 3.
  • the transformed cells were plated on L agar containing 100 ⁇ g/ml ampicillin, and the ampicillin-resistant transformants were screened by restriction enzyme analysis of their plasmid DNA to identify the desired E. coli K12 HB101/pRC colonies.
  • Plasmid pRC DNA was obtained from the E. coli K12 HB101/pRC transformants in substantial accordance with the procedure of Example 1.
  • plasmid pRC DNA in 2 ⁇ l of TE buffer were added to 2 ⁇ l of 10X Hin dIII buffer and 16 ⁇ l of H2O.
  • About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Hin dIII were added to the solution of plasmid pRC DNA, and the resulting reaction was incubated at 37°C for two hours.
  • the Hin dIII-digested plasmid pRC DNA was precipitated with ethanol and resuspended in 2 ⁇ l of 10X Bam HI buffer and 16 ⁇ l of H2O.
  • About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Bam HI were added to the solution of Hin dIII-digested plasmid pRC DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Bam HI- Hin dIII-digested plasmid pRC DNA was extracted once with phenol and then twice with chloroform. The DNA was precipitated with ethanol and resuspended in 3 ⁇ l of 10X ligase buffer and 20 ⁇ l of H2O. The ⁇ 4 ⁇ g (in ⁇ 5 ⁇ l of TE buffer) of ⁇ 2.0 kb Hin dIII- Bam HI restriction fragment of plasmid pTPA102 were then added to the solution of Bam HI- Hin dIII-­digested plasmid pRC DNA. About 2 ⁇ l ( ⁇ 1000 units) of T4 DNA ligase were added to the mixture of DNA, and the resulting reaction was incubated at 16°C for 2 hours. The ligated DNA constituted the desired plasmid pTPA103 DNA.
  • the ligated DNA was digested with restriction enzyme Nco I, which cuts plasmid pRC but not plasmid pTPA103.
  • Nco I restriction enzyme
  • About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Nco I were added to the solution of DNA, and the resulting reaction was incu­bated at 37°C for 2 hours.
  • E. coli K12 RV308 (NRRL B-15624).
  • E. coli K12 RV308 cells were made competent and transformed in substantial accordance with the procedure of Example 3.
  • the transformation mixture was plated on L agar containing 100 ⁇ g/ml ampicillin.
  • the ampicillin-­resistant transformants were tested for sensitivity to kanamycin, for though plasmid pRC confers kanamycin resistance, plasmid pTPA103 does not.
  • the ampicillin-­resistant, kanamycin-sensitive transformants were then used to prepare plasmid DNA, and the plasmid DNA was examined by restriction enzyme analysis to identify the E. coli K12 RV308/pTPA103 transformants.
  • Plasmid pTPA103 DNA was isolated from the E. coli K12 RV308/pTPA103 cells in substantial accordance with the procedure of Example 1.
  • plasmid pTPA103 DNA in 1 ⁇ l of TE buffer was added to 2 ⁇ l of 10X Bgl II buffer and 16 ⁇ l of H2O.
  • About 1 ⁇ l ( ⁇ 5 units) of restriction enzyme Bgl II was added to the solution of plasmid pTPA103 DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Bgl II-digested plasmid pTPA103 DNA was precipitated with ethanol and resuspended in 5 ⁇ l of 10X Klenow buffer and 44 ⁇ l of H2O.
  • Klenow enzyme ( ⁇ 1 unit) was added to the solution of Bgl II-digested plasmid pTPA103 DNA, and the resulting reaction was incubated at 16°C for 2 hours.
  • the Klenow-treated, Bgl II-digested plasmid pTPA103 DNA was precipitated with ethanol and resuspended in 3 ⁇ l of 10X ligase buffer and 22 ⁇ l of H2O.
  • the ligated DNA constituted plasmid pTPA103der Nde I, which is substantially similar to plasmid pTPA103, except plasmid pTPA103der Nde I has an Nde I recognition sequence where plasmid pTPA103 has a Bgl II recognition sequence.
  • the ligated DNA was used to transform E. coli K12 RV3O8 competent cells in substantial accordance with the procedure described in Example 3.
  • the trans­formed cells were plated on L-agar containing ampi­cillin, and the E. coli K12 RV308/pTPA103der Nde I transformants were identified by restriction enzyme analysis of their plasmid DNA. Plasmid pTPA103der Nde I DNA was isolated from the transformants for use in subsequent constructions in substantial accordance with the procedure of Example 1.
  • About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Ava II were added to the DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Ava II-digested DNA was loaded onto an agarose gel and electrophoresed until the ⁇ 1.4 kb restriction fragment was separated from the other digestion products.
  • Hpa I linkers of sequence were kinased in substantial accordance with the pro­cedure of Example 2.
  • About 10 ⁇ l of the kinased linkers were added to the solution of Klenow-treated, ⁇ 1.4 kb Ava II restriction fragment of plasmid pTPA103der Nde I together with 2 ⁇ l ( ⁇ 1000 units) of T4 DNA ligase and 1 ⁇ l ( ⁇ 1 unit) of T4 RNA ligase, and the resulting reaction was incubated at 16°C overnight.
  • the ligated DNA was extracted once with phenol, extracted twice with chloroform, precipitated with ethanol, and resuspended in 2 ⁇ l of 10X Eco RI buffer and 16 ⁇ l of H2O. About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Eco RI were added to the solution of DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Eco RI-digested DNA was extracted once with phenol, extracted twice with chloroform, precipitated with ethanol, and resuspended in 3 ⁇ l of 10X ligase buffer and 20 ⁇ l of H2O.
  • the fragment which is about 770 bp in size and encodes the trp PO and the amino-terminus of TPA, thus prepared had one Eco RI-­compatible end and one blunt end and was ligated into Eco RI- Sma I-digested plasmid pUC19 to form plasmid pUC19TPAFE.
  • plasmid pUC19 available from Bethesda Research Laboratories
  • 16 ⁇ l of H2O 16.
  • 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Sma I were added to the solution of DNA, and the resulting reaction was incubated at 25°C for 2 hours.
  • the Sma I-digested plasmid pUC19 DNA was precipitated with ethanol, collected by centrifugation, and resus­pended in 2 ⁇ l of 10X Eco RI buffer and 16 ⁇ l of H2O. About 2 ⁇ l ( ⁇ 10 units) of restriction enzyme Eco RI were added to the solution of Sma I-digested plasmid pUC19 DNA, and the resulting reaction was incubated at 37°C for 2 hours. The Eco RI- Sma I-digested plasmid pUC19 DNA was extracted once with phenol, extracted twice with chloroform, and resuspended in 5 ⁇ l of TE buffer.
  • the Eco RI- Sma I-digested plasmid pUC19 DNA was added to the solution containing the ⁇ 770 bp Eco RI-­blunt end restriction fragment derived from plasmid pTPA103der Nde I. About 2 ⁇ l ( ⁇ 1000 units) of T4 DNA ligase were added to the mixture of DNA, and the resulting reaction was incubated at 16°C overnight. The ligated DNA constituted the desired plasmid pUC19TPAFE.
  • a restriction site and function map of plasmid pUC19TPAFE is presented in Figure 9 of the accompanying drawings.
  • the multiple-cloning site of plasmid pUC19 which comprises the Eco RI and Sma I recognition sequences utilized in the construction of plasmid pUC19TPAFE, is located within the coding sequence for the lac Z ⁇ fragment.
  • Expression of the lac Z ⁇ fragment in cells that contain the lac Z ⁇ M15 mutation, a mutation in the lac Z gene that encodes ⁇ -galactosidase allows those cells to express a functional ⁇ -galactosidase molecule and thus allows those cells to hydrolyze X-Gal (5-­bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside), a colorless compound, to its indigo-colored hydrolysis product.
  • Insertion of DNA into the multiple-cloning site of plasmid pUC19 interrupts the coding sequence for the lac Z ⁇ fragment, and cells with the lac Z ⁇ M15 muta­tion that host such a plasmid are unable to hydrolyze X-Gal.
  • the ligated DNA that constituted plasmid pUC19TPAFE was used to transform E. coli K12 RR1 ⁇ M15 (NRRL B-15440) cells made competent for transformation in substantial accordance with the procedure of Example 3.
  • the transformed cells were plated on L agar containing 100 ⁇ g/ml ampicillin; 40 ⁇ g/ml X-Gal; and 1 mM IPTG. Colonies that failed to exhibit the indigo color were subcultured and used to prepare plasmid DNA; the E. coli K12 RR1 ⁇ M15/pUC19TPAFE transformants were identified by restriction enzyme analysis of their plasmid DNA. Plasmid pUC19TPAFE DNA was isolated from the E. coli K12 RR1 ⁇ M15/pUC19TPAFE cells for use in subsequent constructions in substantial accordance with the procedure of Example 1.
  • H2O H2O
  • About 3 ⁇ l ( ⁇ 6 units) of restriction enzyme Hpa I were added to the solution of plasmid pUC19TPAFE DNA, and the resulting reaction was incu­bated at 37°C for 20 minutes; the short reaction period was designed to yield a partial Hpa I digest.
  • 1X Bam HI buffer 150 mM NaCl
  • 10 mM Tris-HCl, pH 8.0
  • 10 mM MgCl2 10 mM MgCl2
  • the Bam HI-partially- Hpa I-digested plasmid pUC19TPAFE DNA was concentrated by ethanol precipita­tion, loaded onto a 1.5% agarose gel, and the ⁇ 3.42 kb Hpa I- Bam HI restriction fragment that comprises the replicon, ⁇ -lactamase gene, and all of the TPA-encoding DNA of plasmid pUCATPAFE was isolated from the gel by cutting out the segment of the gel that contained the desired fragment, freezing the segment, and then squeezing the liquid from the segment. The DNA was precipitated from the liquid by an ethanol precipita­tion. About 1 ⁇ g of the desired fragment was obtained and suspended in 20 ⁇ l of TE buffer.
  • the reaction volume was adjusted to 150 ⁇ l of 1X Bam HI buffer, and about l ⁇ l ( ⁇ 16 units) of restriction enzyme Bam HI was added to the mixture, which was then incubated at 37°C for 90 minutes.
  • the DNA was precipitated with ethanol, collected by centrifugation, and resuspended in preparation for electrophoresis.
  • the Sca I- Bam HI-digested plasmid pTPA103 DNA was loaded onto a 1.5% agarose gel and electrophoresed until the ⁇ 1.015 kb Sca I- Bam HI restriction fragment was separated from the other digestion products.
  • the ⁇ 1.015 Sca I- Bam HI restriction fragment that comprises the TPA carboxy-terminus-encoding DNA of plasmid pTPA103 was isolated from the gel; about 0.5 ⁇ g of the desired fragment were obtained and dissolved in 20 ⁇ l of glass-distilled H2O.
  • the ligated DNA was used to transform E. coli K12 JM105 (available from BRL) that were made competent for transformation in substantial accordance with the procedure of Example 3, except that 50 mM CaCl2 was used in the procedure.
  • the transformed cells were plated on BHI (Difco Laboratories, Detroit, MI) containing 100 ⁇ g/ml ampicillin, and the E. coli K12 JM105/pBW25 transformants were identified by restriction enzyme analysis of their plasmid DNA. Digestion of plasmid pBW25 with restriction enzyme Eco RI yields ⁇ 3.38 kb and ⁇ 1.08 kb restriction fragments. Plasmid pBW25 is prepared for use in subsequent constructions in sub­stantial accordance with the procedure of Example 1.
  • plasmid pBW25 in 10 ⁇ l of glass-distilled H2O were added to about 10 ⁇ l of 10X Hin dIII reaction buffer and 80 ⁇ l of H2O.
  • About 1 ⁇ l ( ⁇ 20 units) of restriction enzyme Hin dIII was added to the solution of plasmid pBW25 DNA, and the resulting reaction was incubated at 37°C for 90 minutes.
  • About 3 ⁇ l ( ⁇ 24 units) of restriction enzyme Eco RI and 10 ⁇ l of IM Tris ⁇ HCl, pH 7.6, were added to the solution of Hin dIII-digested plasmid pBW25 DNA, and the resulting reaction was incubated at 37°C for 90 minutes.
  • the Eco RI- Hin dIII-digested plasmid pBW25 DNA was concen­trated by ethanol precipitation, loaded onto a 1.5% agarose gel, and electrophoresed until the ⁇ 810 bp Eco RI- Hin dIII restriction fragment was separated from the other digestion products. About 0.5 ⁇ g of the ⁇ 810 bp Eco RI- Hin dIII restriction fragment was isolated from the gel, prepared for ligation, and resuspended in 20 ⁇ l of glass-distilled H2O.
  • the Hin dIII- Eco RI-digested M13mp8 DNA was collected by ethanol precipitation, resuspended in preparation for agarose gel electrophoresis, and the large restriction fragment isolated by gel electro­phoresis. About 1 ⁇ g of the large Eco RI- Hin dIII restriction fragment of M13mp8 was obtained and suspended in 20 ⁇ l of glass-distilled H2O.
  • E. coli JM103 cells available from BRL, were made competent and transfected with the ligation mix in substantial accordance with the procedure described in the BRL M13 Cloning/'Dideoxy' Sequencing Instruction Manual, except that the amount of DNA used per trans­fection was varied.
  • Recombinant plaques were identified by insertional inactivation of the ⁇ -galactosidase ⁇ -fragment-encoding gene, which results in the loss of the ability to cleave X-gal to its indigo-colored cleavage product.
  • six white plaques were picked into 2.5 ml of L broth, to which was added 0.4 ml of E.
  • the desired phage contained the ⁇ 810 bp Eco RI- Hin dIII restriction fragment of plasmid pBW25 ligated to the ⁇ 7.2 kb Eco RI- Hin dIII restriction fragment of M13mp8.
  • the synthetic oligodeoxyribonucleotide primer used to delete the coding sequence for amino acid residues 87 through 261 of native TPA was 5′-GGGAAGTGCTGTGAAATATCCACCTGCGGCCTGAGA-3′.
  • the resulting mutagenesis mix was used to transfect E. coli K12 JM103 in substantial accordance with the infection procedure described above. Desired mutants were identified by restriction enzyme analysis of RF DNA and by Maxam and Gilbert DNA sequencing. The desired mutant, which had the coding sequence for amino acid residues 87 through 261 of native TPA deleted, was designated pM8BW27.
  • plasmid pBW28 a variety of DNA fragments are needed. The first of these fragments was obtained by adding ⁇ 20 ⁇ g of RF pM8BW27 DNA in 20 ⁇ l of glass-distilled H2O to 10 ⁇ l of 10X Nde I buffer and 60 ⁇ l of H2O. About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Nde I were added to the mixture of plasmid pM8BW27 DNA, and the resulting reaction was incubated at 37°C for two hours.
  • the Nde I-digested plasmid pM8BW27 DNA was precipitated with ethanol, collected by centrifugation, and resuspended in 10 ⁇ l of 10X Eco RI buffer and 90 ⁇ l of H2O. About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Eco RI were added to the solution of Nde I-digested plasmid pM8BW27 DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Eco RI- Nde I-digested plasmid pM8BW27 DNA was electrophoresed on an agarose gel until the ⁇ 560 bp Nde I- Eco RI restriction fragment, which contains the portion of TPA coding sequence that spans the site of deletion, was separated from the other digestion products.
  • the ⁇ 560 bp Nde I- Eco RI restriction fragment was isolated from the gel; about 0.5 ⁇ g of the desired fragment was obtained and suspended in 20 ⁇ l of glass-distilled H2O.
  • the linker has the following structure:
  • the third fragment needed to construct plasmid pBW28 was prepared by adding ⁇ 20 ⁇ g of plasmid pTPA103 in 20 ⁇ l of TE buffer to 10 ⁇ l of 10X Bam HI buffer and 60 ⁇ l of H2O. About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Bam HI were added to the solution of plasmid pTPA103 DNA, and the resulting reaction was incubated at 37°C for 2 hours. The Bam HI-digested plasmid pTPA103 DNA was precipitated with ethanol, collected by centri­fugation, and resuspended in 10 ⁇ l of 10X Eco RI buffer and 80 ⁇ l of H2O.
  • plasmid pBW28 The final fragment necessary to construct plasmid pBW28 was isolated from plasmid pL110, the construction of which was disclosed in Example 9.
  • About 25 ⁇ g of plasmid pL110 in 25 ⁇ l of TE buffer were added to 10 ⁇ l of 10X Xba I buffer (0.5 M NaCl; 60 mM Tris-HCl, pH 7.9; 60 mM MgCl2; and 1 mg/ml BSA) and 55 ⁇ l of H2O.
  • About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Xba I were added to the solution of plasmid pL110 DNA, and the resulting reaction was incubated at 37°C for 2 hours.
  • the Xba I-digested plasmid pL110 DNA was precipitated with ethanol, collected by centrifugation, and resuspended in 10 ⁇ l of 10X Bam HI buffer and 89 ⁇ l of H2O. About 1 ⁇ l ( ⁇ 5 units) of restriction enzyme Bam HI was added to the solution of Xba I-digested plasmid pL110 DNA, and the resulting reaction was incubated at 37°C for 30 minutes to obtain a partial Bam HI digest.
  • the Xba I-partially- Bam HI-digested plasmid pL110 DNA was loaded onto an agarose gel and electrophoresed until the ⁇ 6.0 kb Xba I- Bam HI fragment was clearly separated from the other digestion products.
  • the ⁇ 6.0 kb restriction fragment was isolated from the gel; about 0.5 ⁇ g of the ⁇ 6.0 kb Xba I- Bam HI restriction fragment was obtained and suspended in about 40 ⁇ l of glass-distilled H2O.
  • This ⁇ 6.0 kb Xba I- Bam HI restriction fragment comprises all of plasmid pL110 except the EK-BGH-encoding DNA.
  • plasmid pBW28 To construct plasmid pBW28, the following fragments are mixed together: about 0.1 ⁇ g ( ⁇ 8 ⁇ l) of the ⁇ 6.0 kb Bam HI- Xba I restriction fragment of plasmid pL110; about 0.05 ⁇ g ( ⁇ 2 ⁇ l) of the ⁇ 560 bp Nde I- Eco RI restriction fragment of plasmid pM8BW27; about 0.1 ⁇ g ( ⁇ 2 ⁇ l) of the ⁇ 689 bp Eco RI- Bam HI restriction fragment of plasmid pTPA103; and about 0.02 ⁇ g ( ⁇ 1 ⁇ l) of the ⁇ 45 bp Xba I- Nde I synthetic linker.
  • the ligated DNA was used to transform E. coli K12 MM294 (NRRL B-15625) made competent in substantial accordance with the procedure of Example 3, except that 50 mM CaCl2 was used in the procedure. Due to the presence of the lambda pl promoter and the gene encoding the temperature-sensitive lambda pl repressor on plasmid pBW28, the transformation procedure and culturing of transformants were varied somewhat. The cells were not exposed to temperatures greater than 32°C during trans­formation and subsequent culturing. The desired E. coli K12 MM294/pBW28 transformants were identified by their tetracycline-resistant, ampicillin-sensitive phenotype and by restriction enzyme analysis of their plasmid DNA.
  • plasmid pSV2- ⁇ -globin DNA (NRRL B-15928) were dissolved in 10 ⁇ l 10X Hin dIII reaction buffer, 5 ⁇ l ( ⁇ 50 units) restriction enzyme Hin dIII, and 85 ⁇ l H2O, and the reaction was placed at 37°C for 2 hours. The reaction mixture was then made 0.15 M in LiCl, and after the addition of 2.5 volumes of ethanol and incubation in a dry ice-ethanol bath, the DNA was pelleted by centrifugation.
  • the DNA pellet was dissolved in 10 ⁇ l 10X Bgl II buffer, 5 ⁇ l ( ⁇ 50 units) restriction enzyme Bgl II, and 85 ⁇ l H2O, and the reaction was placed at 37°C for two hours. After the Bgl II digestion, the reaction mixture was loaded onto a 0.85% agarose gel, and the fragments were separated by electrophoresis. The gel was visualized using ethidium bromide and ultraviolet light, and the band containing the desired ⁇ 4.2 kb Hin dIII- Bgl II fragment was excised from the gel as previously described.
  • the pellet was resuspended in 10 ⁇ l of H2O and constituted ⁇ 5 ⁇ g of the desired ⁇ 4.2 kb Hin dIII- Bgl II restriction fragment of plasmid pSV2- ⁇ -­globin.
  • the ⁇ 2.0 kb Hin dIII- Bam HI restriction fragment of plasmid pTPA103 that encodes TPA was isolated from plasmid pTPA103 in substantial accordance with the foregoing teaching.
  • About 5 ⁇ g of the ⁇ 2.0 kb Hin dIII- Bam HI restriction fragment of plasmid pTPA103 were obtained, suspended in 10 ⁇ l of H2O, and stored at -20°C.
  • Plasmid DNA was obtained from the E. coli K12 RR1/pTPA301 transformants in substantial accordance with the procedure of Example 1.
  • a restriction site and function map of plasmid pTPA301 is presented in Figure 10 of the accompanying drawings.
  • Plasmid pSV2-dhfr comprises a dihydrofalate reductase (dhfr) gene useful for selection of trans­formed eukaryotic cells and amplification of DNA covalently linked to the dhfr gene.
  • dhfr dihydrofalate reductase
  • 5 ⁇ l of 5 mM ATP 5 ⁇ l of BSA (1 mg/
  • the DNA was extracted with phenol:chloroform, then extracted with chloroform:isoamyl alcohol, ethanol precipitated, and resuspended in 20 ⁇ l H2O.
  • Plasmid pTPA303 was used to transform E. coli K12 RR1 (NRRL B-15210), and the resulting E. coli K12 RR1/pTPA303 transformants were identified by their ampicillin-resistant phenotype and by restriction enzyme analysis of their plasmid DNA. Plasmid pTPA303 was isolated from the transformants in substantial accord­ance with the procedure of Example 1. A restriction site and function map of plasmid pTPA303 is presented in Figure 10 of the accompanying drawings.
  • the Eco RI- Bgl II-digested DNA is loaded onto an agarose gel and electrophoresed until the ⁇ 2.7 kb Eco RI- Bgl II restriction fragment is separated from the other digestion products, and then, the ⁇ 2.7 kb fragment is isolated and prepared for ligation.
  • plasmid pTPA303 was double-­digested with Hin dIII and Eco RI restriction enzymes, and the ⁇ 2340 bp Eco RI- Hin dIII restriction fragment that comprises the dhfr gene was isolated and recovered.
  • Plasmid pTPA303 was double digested with Hin dIII and Sst I restriction enzymes in IX Hin dIII buffer. The ⁇ 1.7 kb fragment was isolated from the gel and prepared for ligation.
  • Plasmid pBW32 DNA was obtained from the E. coli K12 MM294/pBW32 transformants in substantial accordance with the procedure of Example 1.
  • a restriction site and function map of plasmid pBW32 is presented in Figure 10 of the accompanying drawings.
  • plasmid pBW32 in 20 ⁇ l of TE buffer were added to 10 ⁇ l of 10X Bam HI buffer and 60 ⁇ l of H2O.
  • About 10 ⁇ l ( ⁇ 50 units) of restriction enzyme Bam HI were added to the solution of plasmid pBW32 DNA, and the resulting reaction was incubated at 37°C for two hours.
  • the Bam HI-digested plasmid pBW32 DNA was pre­cipitated with ethanol, collected by centrifugation, and resuspended in 5 ⁇ l of 10X Klenow buffer, 45 ⁇ l of H2O, and 2 ⁇ l ( ⁇ 100 units) of Klenow enzyme.
  • the reaction was incubated at 16°C for 30 minutes; then, the reaction mixture was loaded onto an agarose gel and electro­phoresed until the digestion products were clearly separated.
  • the ⁇ 1.9 kb Klenow-treated, Bam HI restric­tion fragment of plasmid pBW32 that comprises the dhfr gene was isolated from the gel and prepared for ligation in substantial accordance with the procedure of Example 12A. About 4 ⁇ g of the desired fragment were obtained and suspended in 5 ⁇ l of TE buffer.
  • plasmid pLPChyg1 in 100 ⁇ l of TE buffer were added to 15 ⁇ l of 10X Eco RI buffer and 30 ⁇ l of H2O.
  • About 5 ⁇ l ( ⁇ 50 units) of restriction enzyme Eco RI were added to the solution of plasmid pLPChyg1 DNA, and the resulting reaction was incubated at 37°C for about 10 minutes. The short reaction time was calculated to produce a partial Eco RI digestion.
  • Plasmid pLPChyg1 has two Eco RI restriction sites, one of which is within the coding sequence of the hygromycin resistance-conferring (HmR) gene, and it was desired to insert the dhfr-gene-containing restriction fragment into the Eco RI site of plasmid pLPChyg1 that is not in the HmR gene.
  • the partially- Eco RI-digested plasmid pLPChyg1 DNA was loaded onto an agarose gel and electrophoresed until the singly-cut plasmid pLPChyg1 DNA was separated from uncut plasmid DNA and the other digestion products. The singly-cut DNA was isolated from the gel and prepared for ligation in substantial accordance with the procedure of Example 12A.
  • the ligated DNA was used to transform E. coli K12 HB101 cells made competent for transformation in substantial accordance with the procedure of Example 3.
  • the transformed cells were plated onto L agar containing 100 ⁇ g/ml ampicillin, and the ampicillin-resistant transformants were analyzed by restriction enzyme analysis of their plasmid DNA to identify the E. coli K12 HB101/pLPChd1 and E. coli K12 HB101/pLPChd2 trans­formants.
  • plasmid pLPChd1 has been designated plasmid pLChd.
  • a restric­tion site and function map of plasmid pLPChd is pre­sented in Figure 11 of the accompanying drawings. Plasmid pLPChd1 and plasmid pLPChd2 DNA were isolated from the appropriate transformants in substantial accordance with the procedure of Example 1.
  • E. coli K12 GM48 (NRRL B-15725) lacks a functional dam methylase and so is a suitable host to use for the purpose of preparing plasmid pLPChd1 DNA for use as starting material in the construction of plasmid phd.
  • E. coli K12 GM48 cells were cultured and made competent for transformation, and plasmid pLPChyg1 was used to transform the E. coli K12 GM48 cells in substantial accordance with the procedure of Example 3.
  • the transformed cells were plated on L agar containing ampicillin, and once the ampicillin-resistant, E. coli K12 GM48/pLPChd1 transformants had formed colonies, one such colony was used to prepare plasmid pLPChd1 DNA in substantial accordance with the procedure of Example 1. About 1 mg of plasmid pLPChd1 DNA was obtained and suspended in about 1 ml of TE buffer.
  • Plasmid phd results from the deletion of the extra Bcl I linkers that attached during the construction of plasmid pLPcat and the two adjacent Bcl I restriction fragments of a total size of about 1.45 kb from plasmid pLPChd1.
  • Plasmid phd facilitates the expression of any DNA sequence from the BK virus enhancer-adenovirus late promoter of the present invention, because the DNA to be expressed can be readily inserted in the correct position for expression at the single Bcl I site on plasmid phd.
  • the ligated DNA was used to transform E. coli K12 GM48 in substantial accordance with the procedure of Example 3.
  • the transformed cells were plated on L agar containing ampicillin, and the ampicillin-resistant E. coli K12 GM48/phd transformants were identified by restriction enzyme analysis of their plasmid DNA.
  • Plasmid pGKC2310 contains the entire, full-­length cDNA which encodes the light chain of murine monoclonal antibody KS1/4.
  • E. coli K12 MM294/pGKC2310 can be obtained from the Northern Regional Research Laboratory under the accession number NRRL B-18356.
  • a restriction site and function map of plasmid pGKC2310 is presented in Figure 12 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except that the temperature of incubation is 37°C.
  • plasmid pGKC2310 About 5 ⁇ g of plasmid pGKC2310 are digested with restriction enzyme Eco RI in substantial accordance with the teaching of Example 8, then treated with Klenow in substantial accordance with the teaching of Example 5.
  • Bam HI linkers purchased from New England Biolabs
  • the DNA is electrophoresed through an agarose gel and purified in substantial accordance with the teaching of Example 12.
  • the desired ⁇ 1.1 kb fragment is ethanol precipitated and resuspended in 5 ⁇ l TE.
  • plasmid phd About 1 ⁇ g of plasmid phd was digested with restriction enzyme Bcl I in substantial accordance with the teaching of Example 18, then electrophoresed and purified in substantial accordance with the teaching of Example 12.
  • the ⁇ 1.1 kb Eco RI-digested, Klenow-­treated, linker-modified DNA fragment of plasmid pGKC2310 is then ligated into the Bcl I-digested plasmid phd in substantial accordance with the teaching of Example 2.
  • This ligation mixture is then transformed into E. coli cells in substantial accordance with the teaching of Example 3.
  • the plasmids are then isolated from the cells in substantial accordance with the teaching of Example 1, and the plasmid containing the correct orientation is designated plasmid pL-KSL.
  • Plasmid pG2A52 contains the entire, full-­length cDNA which encodes the heavy chain of murine monoclonal antibody KS1/4.
  • E. coli K12 MM294/pG2A52 can be obtained from the Northern Research Laboratory under the accession number NRRL B-18357.
  • a restriction site and function map of plasmid pG2A52 is presented in Figure 12 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except that the temperature of incubation is 37°C.
  • plasmid PG2A52 About 5 ⁇ g of plasmid PG2A52 are digested with restriction enzyme Eco RI in substantial accordance with the teaching of Example 8, then treated with Klenow in substantial accordance with the teaching of Example 5.
  • Bam HI linkers purchased from New England Biolabs
  • the DNA is electrophoresed through an agarose gel and purified in substantial accordance with the teaching of Example 12.
  • the desired ⁇ 1.6 kb fragment is ethanol precipitated and resuspended in 5 ⁇ l TE.
  • plasmid pH-KS About 1 ⁇ g of plasmid phd was digested with restriction enzyme Bcl I in substantial accordance with the teaching of Example 18, then electrophoresed and purified in substantial accordance with the teaching of Example 12.
  • the ⁇ 1.6 kb Eco RI-digested, Klenow-treated, linker-modified DNA fragment of plasmid pG2A52 is then ligated into the Bcl I-digested plasmid phd in substantial accordance with the teaching of Example 2.
  • This ligation mixture is then transformed into E. coli cells in substantial accordance with the teaching of Example 3.
  • the plasmids are then isolated from the cells in substantial accordance with the teaching of Example 1 and the plasmid containing the correct orientation is designated plasmid pH-KS.
  • Plasmid pCHKC2-18 contains a cDNA fragment which encodes the light chain variable region of murine monoclonal antibody KS1/4 joined to a genomic DNA fragment which encodes a human light chain constant region.
  • the variable region encoded by plasmid pCHKC2-18 contains a single amino acid alteration from the naturally-occurring variable region of monoclonal antibody KS1/4.
  • the carboxy-terminal amino acid in the wild-type variable region is an arginine residue, whereas said residue is a glycine residue in the protein encoded by plasmid pCHKC2-18.
  • E. coli K12 DH5/pCHK2-18 can be obtained from the Northern Regional Research Laboratory under the accession number NRRL B-18359.
  • a restriction site and function map of plasmid pCHKC2-18 is present in Figure 13 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except that the temperature of incubation is 37°C.
  • plasmid pCHKC2-18 was digested in substantial accordance with the teaching of Example 8, except restriction enzyme Fnu DII and 10X Fnu DII buffer (60 mM NaCl, 60 mM Tris-HCl (pH 7.4) and 60 mM MgCl2) were used.
  • the DNA was next digested using restriction enzyme Hin dIII and 10X Hin dIII buffer (500 mM NaCl, 500 mM Tris-HCl (pH 8.0) and 100 mM MgCl2), then the ⁇ 1.3 kb Fnu DII- Hin dIII fragment was isolated from an agarose gel in substantial accordance with the teaching of Example 12. After ethanol precipitation and resus­pension, the 5′ overhang of the Hin dIII site was filled in using Klenow, in substantial accordance with the teaching of Example 5.
  • plasmid pL-HD The plasmids were then isolated from the cells in substantial accordance with the teaching of Example 1, and the plasmid containing the correct orientation (evidenced by an ⁇ 840 bp Mae II- Stu I frag­ment) was designated plasmid pL-HD.
  • Plasmid pCHKC2-6 contains a cDNA fragment which encodes the light chain variable region of mono­clonal antibody KS1/4 joined to a genomic DNA fragment which encodes a human light chain constant region.
  • the variable region encoded by plasmid pCHKC2-6 contains the same amino acid residue sequence as the naturally-­occurring variable region of monoclonal KS1/4.
  • the DNA which encoded the carboxy-terminal arginine residue contains a CGT codon in plasmid pCHKC2-6, rather than the CGG codon which occurs in the natural sequence.
  • E. coli K12 DH5/pCHKC2-6 can be obtained from the Northern Regional Research Laboratory under the accession number NRRL B-18358.
  • a restriction site and function map of plasmid pCHKC2-6 is presented in Figure 13 of the accompanying drawings.
  • Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except the temperature of incubation is 37°C. Plasmid pCHKC2-6 is then digested with restriction enzymes Fnu DII and Hin dIII, treated with Klenow, then ligated into Bcl I-cut, filled-in plasmid phd in substantial accordance with the teaching of Example 21. The resultant plasmid is then transformed into E. coli , extracted and mapped in substantial accordance with the teaching of Example 21. The plasmid with the correct ⁇ 840 bp Mae II- Stu I fragment is designated plasmid pL-HD2.
  • Plasmid pCH2A5 contains a cDNA fragment which encodes the heavy chain variable region of murine mono­clonal antibody KS1/4 joined to a genomic DNA fragment which encodes a human heavy chain constant region of immunoglobulin IgG1.
  • E. coli K12 MM294/pCH2A5 can be obtained from the Northern Regional Research Labora­tories under the accession number NRRL B-18360.
  • a restriction site and function map of plasmid pCH2A5 is presented in Figure 14 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except the temperature of incubation is 37°C.
  • plasmid pCH2A5 About 10 ⁇ g of plasmid pCH2A5 were cut with restriction enzyme Eco RI in substantial accordance with the teaching of Example 5.
  • the DNA was treated with Klenow and Bam HI linkers (purchased from New England Biolabs) were then added in substantial accordance with the teaching of Example 2, and the DNA was digested with restriction enzyme Bam HI in substantial accordance with the teaching of Example 5.
  • the ⁇ 7.4 kb Bam HI- Eco RI/ Bam HI restriction fragment was then isolated from an agarose gel in substantial accordance with the teaching of Example 12. This fragment was then ligated into the Bcl I digested phd vector (Example 20) in substantial accordance with the teaching of Example 2.
  • the DNA was then transformed into E. coli , reisolated, and the plasmid with the correct orientation (evidenced by an ⁇ 780 bp Mae III- Stu I fragment) was designated plasmid pH1-HD.
  • Plasmid pCH2A5IG2 contains a cDNA fragment which encodes the heavy chain variable region of murine mono­clonal antibody KS1/4 joined to a genomic DNA fragment which encodes a human heavy chain constant region of immunoglobulin IgG2.
  • E. coli K12 DH5/pCH2A5IG2 can be obtained from the Northern Regional Research Labora­tories under the accession number NRRL B-18361.
  • a restriction site and function map of plasmid pCH2A5IG2 is presented in Figure 14 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except the temperature of incubation is 37°C.
  • plasmid pCH2A5IG2 About 10 ⁇ g of plasmid pCH2A5IG2 were cut with restriction enzyme Eco RI in substantial accordance with the teaching of Example 5.
  • the DNA was treated with Klenow and Bam HI linkers (purchased from New England Biolabs) were then added in substantial accordance with the teaching of Example 2, and the DNA was digested with restriction enzyme Bam HI in substantial accordance with the teaching of Example 5.
  • the ⁇ 6.1 kb Bam HI- Eco RI/ Bam HI restriction fragment was then isolated from an agarose gel in substantial accordance with the teaching of Example 12. This fragment was then ligated into the Bcl I digested phd vector (Example 20) in substantial accordance with the teaching of Example 2.
  • the DNA was then transformed into E. coli reisolated, and the plasmid with the correct orientation (evidenced by an ⁇ 780 bp Mae III- Stu I fragment) was designated plasmid pH2-HD.
  • Plasmid pCH2A5IG3 contains a cDNA fragment which encodes the heavy chain variable region of murine mono­clonal antibody KS1/4 joined to a genomic DNA fragment which encodes a human heavy chain constant region of immunoglobulin IgG1.
  • E. coli K12 DH5/pCH2A5IG3 can be obtained from the Northern Regional Research Labora­tories under the accession number NRRL B-18362.
  • a restriction site and function map of plasmid pCH2A5IG3 is presented in Figure 15 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except the temperature of incubation is 37°C.
  • plasmid pCH2A5IG3 were cut with restriction enzyme Eco RI in substantial accordance with the teaching of Example 5.
  • the DNA was treated with Klenow and Bam HI linkers (purchased from New England Biolabs) were then added in substantial accordance with the teaching of Example 2, and the DNA was digested with restriction enzyme Bam HI in substantial accordance with the teaching of Example 5.
  • the ⁇ 7.4 kb Bam HI- Eco RI/ Bam HI restriction fragment was then isolated from an agarose gel in substantial accordance with the teaching of Example 12. This fragment was then ligated into the Bcl I digested phd vector (Example 20) in substantial accordance with the teaching of Example 2.
  • the DNA was then transformed into E. coli , reisolated, and the plasmid with the correct orientation (evidenced by an ⁇ 780 bp Mae III- Stu I fragment) was designated plasmid pH3-HD.
  • Plasmid pCH2A5IG4 contains a cDNA fragment which encodes the heavy chain variable region of murine monoclonal antibody KS1/4 joined to a genomic DNA fragment which encodes a human heavy chain constant region of immunoglobulin IgG4.
  • E. coli K12 DH5/pCH2A5IG4 can be obtained from the Northern Regional Research Laboratories under the accession number NRRL B-18363.
  • a restriction site and function map of plasmid pCH2A5IG4 is presented in Figure 15 of the accompanying drawings. Plasmid DNA is extracted from the culture in substantial accordance with the teaching of Example 1, except the temperature of incubation is 37°C.
  • plasmid pH4-HD About 10 ⁇ g of plasmid pCH2A5IG4 were cut with restriction enzyme Eco RI in substantial accordance with the teaching of Example 5.
  • the DNA was treated with Klenow and Bam HI linkers (purchased from New England Biolabs) were then added in substantial accordance with the teaching of Example 2, and the DNA was digested with restriction enzyme Bam HI in substantial accordance with the teaching of Example 5.
  • the DNA was then transformed into E. coli , reisolated, and the plasmid with the correct orientation (evidenced by an ⁇ 780 bp Mae III- Stu I fragment) was designated plasmid pH4-HD.
  • the present expression vectors contain the BK enhancer described in U.S. Patent Application No. 07/129,028, Attorney Docket X-6606A, filed December 4, 1987, the teaching of which is incorporated herein by reference.
  • the BK enhancer stimulates gene expression in the presence of the E1A gene product.
  • 293 cells constitutively express the E1A gene product, 293 cells are an efficient host for the eukaryotic expression vectors of the present invention.
  • 293 cells are human embryonic kidney cells transformed with adenovirus type 5 (note that any particular type of adenovirus can be used to supply the E1A gene product in the method of the present invention) and are available from the ATCC under the accession number CRL 1573.
  • the expression vectors of the present invention function in a wide variety of host cells, even if the E1A gene product is not present.
  • the E1A gene product can be introduced into a non-E1A-producing cell line either by transformation with a vector that comprises the E1A gene, or with sheared adenovirus DNA, or by infection with adenovirus.
  • 293 cells are obtained from the ATCC under the accession number CRL 1573 in a 25 mm2 flask containing a confluent monolayer of about 5.5 X 106 cells in Eagle's Minimum Essential Medium with 10% heat-inactivated horse serum. The flask is incubated at 37°C; medium is changed twice weekly.
  • the cells are subcultured by removing the medium, rinsing with Hank's Balanced Salts solution (Gibco), adding 0.25% trypsin for 1-2 minutes, rinsing with fresh medium, aspirating, and dispensing into new flasks at a subcultivation ratio of 1:5 or 1:10.
  • Hank's Balanced Salts solution Gibco
  • a one ml sterile plastic pipette with a cotton plug is inserted into the mixing tube that contains the 2X HBS, and bubbles are introduced by blowing while the DNA is being added.
  • the calcium-­phosphate-DNA precipitate is allowed to form without agitation for 30-45 minutes at room temperature.
  • the transformation procedure utilizes a mixture of plasmids: an expression vector that lacks a selectable marker; and an expression vector that comprises a selectable marker that functions in eukaryotic cells. This co-transformation technique allows for the identi­fication of cells that comprise both of the transforming plasmids.
  • hygromycin is added to the growth medium to a final concentration of about 200 to 400 ⁇ g/ml. The cells are then incubated at 37°C for 2-4 weeks with medium changes at 3 to 4 day intervals. The resulting hygromycin-resistant colonies are transferred to individual culture flasks for characterization. The selection of neomycin (G418 is also used in place of neomycin)-resistant colonies is performed in substantial accordance with the selection procedure for hygromycin-resistant cells, except that neomycin is added to a final concentration of 400 ⁇ g/ml rather than hygromycin.
  • 293 cells are dhfr positive, so 293 transformants that contain plasmids comprising the dhfr gene are not selected solely on the basis of the dhfr-positive phenotype, which is the ability to grow in media that lacks hypoxanthine and thymine.
  • Cell lines that do lack a functional dhfr gene and are transformed with dhfr-containing plasmids can be selected for on the basis of the dhfr+ phenotype.
  • dhfr dihydrofolate reductase
  • amplifiable markers such as metallothionein genes, adenosine deaminase genes, or members of the multigene resistance family, exemplified by P-glycoprotein, can be utilized.
  • a selectable marker such as the hygromycin B resistance-conferring gene and then amplify using methotrexate, which cannot be used for selection of murine dhfr-containing plasmids in 293 cells.
  • AV12 Cell line AV12 (ATCC CRL 9595) were trans­formed in substantial accordance with the procedure described for 293 cells. AV12 cells also constitutively express the E1A gene product and are the preferred host for the eukaryotic expression vectors of the present invention. However, unlike 293 cells, AV12 cells were directly selected with methotrexate (200-500 nM) when transformed with a vector containing the murine dhfr gene. To express a heavy chain, it was necessary to transform the AV12 cells with any expression vector which encodes a heavy chain. However, a heavy chain will not be secreted into the supernatent unless the AV12 cells are also co-transformed with a vector encoding a light chain.
  • methotrexate 200-500 nM
  • Light chains will be secreted from host cells after transformation with a vector encoding light chains. In this manner, various com­binations of light and heavy chains were expressed via co-transformation and selection of AV12 cells. To assay for the production of a fully assembled, secreted immunoglobulin, one therefore should assay for the presence of a secreted heavy chain.
  • the methotrexate-resistant transformants obtained in Example 27 are grown on 100 mm2 tissue culture dishes at a density of several hundred cell clones per tissue culture dish. The media is decanted, and the cells are rinsed twice with 5 ml aliquots of Hank's Balanced salt solution (Gibco). A solution of sterile 0.45% agar (Sigma Type 4 agarose, catalogue #A3643, Sigma Chemical Co., P.O. Box 14508, St.
  • Louis, MO 63178 is prepared by mixing 1 ml of 1.8% agar (47°C) with 3 ml of Dulbecco's Modified Eagle's (DME) Salts (Gibco) (37°C), and 2 ml of this 0.45% agar solution are layered over the cells.
  • DME Dulbecco's Modified Eagle's
  • the cells are overlayed with 8 ml of a mixture containing 2 ml of 1.8% agar (47°C), 2 ml of DME salts (37°C), and 4 ml of DME salts with 20% fetal bovine serum (37°C). The cells are then placed in a 37°C incubator.
  • Polyclonal antibody can be prepared by the procedure disclosed in Structural Concepts in Immunology and Immunochemistry by E.A. Kabat, published in 1968 by Holt, Rhinehart, and Winston. Monoclonal antibody, which is also suitable for use in the assay, can be prepared as disclosed in Kohler and Milstein, 1975, Nature, 256:495, or as disclosed in U.S. Patent No. 4,696,895; EPO Pub. No. 205046; Laurell et al ., 1985, FEBS 191(1):75; Suzuki et al ., 1985, J. Biochem. 97:127-138; and EPO Pub. No. 138222.
  • the avidin D and biotinylated horse radish peroxidase (HRP) used in the assay are obtained in a VectastainTM kit (Vector Laboratories, Inc. Biotin is also obtained from Vector Laboratories, Inc.
  • the filters are rinsed four times with PBS at 4°C. Then, avidin D and biotinylated horse radish peroxidase are prepared and added as per the manu­facturer's instructions in the VectastainTM (Vector Laboratories) kit. The filters are incubated with the HRP-conjugated avidin D for 1 hour at 4°C (longer incu­bation times, i.e., overnight, can be used when small amounts of protein are being secreted); then, the filters are rinsed four times with PBS at 4°C.
  • HRP color-development reagent (4-chloro-­1-napthol, Sigma) dissolved in ice-cold 100% methanol are added to 50 ml of PBS and 30 ⁇ l of 30% H2O2. This mixture is added to the nitrocellulose filters, which are incubated at room temperature until the color develops. Colonies secreting the most antibody of the invention will be indicated on the filters not only by earliest appearance of the color but also by darker spots on the filter.
  • the filters are again realigned with the original plates to determine which colonies are associated with which spots on the filter. The colonies secreting the most antibody are then selected and used for production of the antibody.
  • the above assay is merely illustrative of the method of identifying high secreting cell lines.
  • a variety of assay procedures can be successfully employed in the method. For instance, a double-antibody reaction can be employed in which the biotinylated goat anti human heavy chain antibody is replaced with a goat anti-human heavy chain (IgG) and a biotinylated anti-goat IgG antibody.
  • IgG goat anti-human heavy chain

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EP1247865A3 (de) * 1990-10-17 2003-06-04 The Wellcome Foundation Limited Antikörper und ihre therapeutische Verwendung
EP1383785A2 (de) * 2001-05-03 2004-01-28 MERCK PATENT GmbH "rekombinanter, tumorspezifischer antikörper und dessen verwendung"

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US4975369A (en) 1990-12-04
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NZ228801A (en) 1991-05-28
AU3318989A (en) 1989-10-26
AU621600B2 (en) 1992-03-19
DK187889D0 (da) 1989-04-19

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